A court spokesman says Khieu Samphan was brought before investigating judges of the U.N.-assisted tribunal Friday and charged.
On Wednesday, the tribunal charged two other defendants with genocide for the first time: Nuon Chea, the group's top ideologist and former foreign minister Ieng Sary.
All three have been charged with involvement in the deaths of members of the country's ethnic Cham and Vietnamese communities.
Some Chams, who are mostly Muslims, were among the few Cambodians to actively resist Khmer Rouge rule. The Khmer Rouge brutally suppressed the rebellions in several villages.
All three are also facing charges of war crimes and crimes against humanity, as well as homicide and torture for their involvement in the deaths of 1.7 million Cambodians between 1975 and 1979.
Although many Cambodians feel their people suffered a genocide, it is difficult for prosecutors to apply the charge to the mass killing.
The United Nations defines genocide as "acts committed with intent to destroy a national, ethnical, racial or religious group."
So far, three of five former senior Khmer Rouge members facing prosecution for their involvement in the brutal ultra-communist regime have been formally charged in court.
Prison chief Kaing Guek Eav, known as Duch, was the first defendant to be tried. He faces life imprisonment if found guilty of crimes against humanity, war crimes, torture and murder. A verdict is expected some time early next year.
Duch commanded the S-21 prison in Phnom Penh, where about 16,000 people were tortured and taken away to be killed.
The Khmer Rouge aimed to wipe out Cambodia's social classes and create a self-sustaining, agrarian society.
They emptied Cambodia's cities, killed anyone who appeared educated or elite, and forced the rest of the population to work in rural labor camps. Vietnamese forces pushed the group from power in 1979.