The fresh FIR was registered by the probe agency after the Special Investigation Team, probing the case, gave its complaint to the CBI on December 15.
All the 20 policemen have been charged with murder and destruction of evidence, a senior CBI officer said.
The Gujarat High Court had on December 1 directed the CBI to take over further probe in the case in which 19-year-old Ishrat, Javed Sheikh alias Pranesh Pillai, Amjad Ali Rana and Zeeshan Johar were killed in an encounter in Ahmedabad.
The directives came as the Special Investigative Team constituted by the High Court had concluded last month that the encounter was staged by police.
A judicial inquiry report by metropolitan magistrate SP Tamang report had alleged that 21 police officers, including then crime branch chief JCP PP Pande, suspended DIG DG Vanzara, the then ACP GL Singhal and ACP NK Amin were involved in the conspiracy regarding the encounter.
Vanzara and Amin are also accused in the fake encounter killing of alleged gangster Sohrabuddin Sheikh and in the murder of his wife Kausar Bi and are at present in jail.
The High Court had ordered CBI to also probe the claims made by the state police after the encounter that Ishrat and the other three persons were LeT terrorists on a mission to kill Gujarat Chief Minister Narendra Modi.