"Within months, the 501st gained a well deserved reputation as 'Vader's Fist.'"

RISE OF THE GALACTIC EMPIRE

As the battle was winding down, a group of clones from the 501st was discreetly transferred to Coruscant, in preparation for Order 66 -a top-secret order given by Chancellor Palpatine which stated that the Jedi had rebelled against the Republic, and needed to be eliminated. On Coruscant, the 501st rendezvoused with the Sith Lord Darth Vader, formerly known as Anakin Skywalker, and were given their orders: destroy the Jedi. Though they appreciated the Jedi, their loyalty lay first with Palpatine, as all clone troopers had been taught to obey the Supreme Chancellor first and foremost. Vader led them into the Jedi Temple, ready for battle. Though some held quiet doubts about what they were commanded to do, years of psychological programming and conditioning prevented any of them from resisting such an extreme order.

With their inability to resist morally-questionable orders, the 501st Legion steadily marched behind Darth Vader and towards the main entrance of the Jedi Temple. Then they carried out Order 66 to the letter by attacking every Jedi in their path, from masters and knights to padawans and younglings. With most of the senior members of the Jedi Order scattered throughout the galaxy on various fronts, the Temple's remaining occupants were left in a hopeless position as Vader and the 501st clone troopers turned the Temple into a scene of slaughter and mayhem. Though the 501st suffered their own fair share of casualties, and even though several Jedi were able to escape from the doomed battle, the clones' superb training and Vader's relentless onslaught resulted in the deaths of almost every Jedi within the Temple. Meanwhile, Palpatine was transmitting Order 66 around the galaxy to different clone commanders. Nearly all obeyed their orders without question, killing their unsuspecting Jedi officers who made the mistake of trusting the clones with their lives.

The Coruscant Security Force tracked a number of suspected Jedi, such as Tru Veld, and called in the 501st for assistance in capturing them. The legion also attacked and killed a number of Jedi on Shinarcan Bridge a couple of hours after the declaration of Order 66, and Jedi Knight Etain Tur-Mukan was killed by a fellow Jedi when she tried to stop him from killing one of the 501st troopers. Following the extermination of the Jedi, Decoy Squad Five was tasked with deceiving any Jedi that returned to the Temple in order to kill them, though the eight-member squad was later killed by Obi-Wan Kenobi and Yoda, who had managed to escape the carnage and return to Coruscant. The 501st troopers that remained behind to occupy the Temple attempted to kill Kenobi and Yoda, only to be wiped out by the two Jedi Masters.