Tamina mentions the "Mughals of the Hindu Kush" when going into Avrat. The Mughal Dynasty did not emerge till the 14th century, almost a millennium later than when the movie is supposed to take place. It spanned almost the entire Indian subcontinent and was by no means limited to the Hindu Kush mountain eange.
During the ostrich races, you can clearly see numbers written in modern Arabic numerals on the backs of the riders. This numbering system was still relatively primitive at the time and would not have been stylized in such a modernly-recognizable form.
As the Persian army is entering the city, a soldier is carrying a pole carrying on the tip the crescent moon of Islam. If the film takes place in 4-6th century as claimed, this is early by about 400 years. (The moon appears to be a 'smile' as in that part of the world, a crescent moon runs from 9 o'clock to 3 o'clock.)
The map in the opening scene shows a Persian Empire stretching to Asia Minor (modern-day Turkey) in the west. Persian rule never again extended this far once Alexander the Great toppled the Achaemenid Dynasty in the 4th century BC. In a featurette on the DVD, the director describes the movie's action as taking place between the 4th and 6th centuries, presumably AD, several centuries after the map would have become obsolete. By that time the Eastern Roman (Byzantine) Empire would have controlled many of the areas shown as western Persia on the map.
When Dastan is on the run from the Persian soldiers in Avrat, he jumps down into a building and onto a railing. When he pulls himself up over the rail, as he plants his feet on the floor, you can see where the leather flaps of his "boots" stop and show his athletic shoes underneath.
When Sheik Amar talks about his last ostrich Anita, he refers to her as "she", when clearly the bird in the picture is a male ostrich. Male ostriches are black with a white tail, while female birds are greyish brown.
When Dastan and Tamina come out of their tent after a massive sand storm, sand has covered everything in sight except for their tent which doesn't have a single spec of sand on it. Also, not a single spec of sand appears to have gotten inside the tent.
At the end of the first battle of the Holy City of Alamut, Tus has a small cut on his right cheek with blood running down his face, but in the next scene, the blood and the cut are gone, and there is no scar.
When Dastan and Tamina are on the sand dune hiding from the patrol, Tamina can be seen holding the dagger when she stands up. Then Dastan approaches her and produces it from behind his back even though he clearly wasn't ever close enough to have taken it from her.
The completely loaded handle of the dagger represents one minute of time that can be rewound. However, when Dastan refills the dagger, the sand shown in the handle does not reflect 60 seconds' worth of time (it's less than a quarter full, not even 15 seconds' worth), yet the full minute is used up in its subsequent activations.
When Dastan presses the jewel on the dagger for the second time, Tamina reaches out to stop him, and is then frozen in time. The next shot, however, shows her fingers in a different position. Additionally, as she is poising to strike, her mouth is open. When she is frozen in time in the next shot, her mouth is closed.
Sheik Amar fights a Hassansin at the sacred temple outside Alamut, and one of their swords is broken off near the hilt (it's not entirely clear whose). In following shots, however, both blades are seen to be completely intact.
After Alamut has been attacked, Tus proposes to Tamina. She refuses but then sees Dastan standing to her right, with the dagger, so she agrees to marry Tus and he lifts her left hand to kiss it, then raises it in the air. But when he lifts her hand, it is her right hand and Dastan is now on her left.
At the very end of the movie the setting sun moves from right to left, which can only be viewed like that on the Southern hemisphere. The Persian Empire was on the Northern hemisphere. (Either the movie makers used a reversed sunrise viewed from the Northern hemisphere, to make it look like a sunset but forgot to mirror the movie or they did it intentionally showing a sunrise going back in time, which is not completely unlikely since the movie revolves around time travel, but really nothing indicates to that)
The letter in the film was written in modern Persian alphabet (derived from Arabic alphabet), that was not used in Persia before Islam.
The replacement of the Pahlavi scripts with the Persian alphabet to write the Persian language was done in 9th-century.
When Tamina is being readied to be presented to King Sharaman her maid wipes the Henna Tattoos revealing clean skin. Henna paste, once dry, leaves distinctive stains on the skin which take up to three weeks to fade.
In the second half of the film, two lines of audio play twice. When explaining the importance of the dagger, Tamina says, "The secret guardian temple outside Alamut is a sanctuary, the one place the dagger can be hidden safely," and then, "Give me back the dagger, so that I can take it there." When they reach the temple, the same audio is used, dubbed in while her back is to the camera. The first line has been trimmed to, "The... sanctuary, the one place the dagger can be hidden safely" while the second line is used exactly. You can tell by the intonation of her voice and pacing of her words.
During his fight with the Hassansin guard, Seso takes several darts to the chest. In the shot immediately afterward, he peeks around a pillar, and his chest is clear. In the following closeup, though no further action has taken place, the darts are seen protruding from his chest.
When Dastan, Tamina and Nizam are are the Glass of Time, Nizam throws Tamina off a cliff and Dastan catches her twice. He catches her once, when the camera angle is from above. Then he catches her again, when the camera angle changes to from below.
In the rebooted time-line after the battle of the Holy City of Alamut, Tus has a two minor wounds on the bridge of his nose and his cheek while confronting Dastan and Nizam. When Tus is speaking to Tamina, both wounds have disappeared.
Dastan can be seen picking up Tus' chain of gold beads twice. First in the scene where Tamina runs to Dastan after discovering the identity of the Hassassin, and again in the scene immediately following.