Winterkorn, who resigned in November 2015 in response to the scandal, said he was not aware of any wrongdoing at the time of his resignation.
An indictment, filed in secret in March, was unsealed on Thursday in US District Court, and it names numerous former executives.
A spokesperson for the US Attorney's office in Detroit said Mr Winterkorn was not in custody.
"Volkswagen continues to cooperate with investigations by the Department of Justice into the conduct of individuals", a VW spokesperson said in an emailed statement. Two people have pleaded guilty in the case.
The indictment alleges that by the summer of 2015, USA regulators threatened to withhold authorization for VW to sell Model Year 2016 diesel vehicles in the United States until VW answered their questions about the discrepancies uncovered by the ICCT study.
VW's false representations to regulators and the public about the ability of VW's supposedly "clean diesel" vehicles to comply with USA emissions requirements affected about 600,000 vehicles across the country.
Federal prosecutors charged Winterkorn with wire fraud, conspiracy to defraud the USA and violating the Clean Air Act.
Volkswagen pleaded guilty previous year to cheating on emissions tests by using software in almost 600,000 of its diesel vehicles in the US and agreed to pay $4.3 billion in penalties. The first count is the most serious as it alleges "Winterkorn conspired with other senior VW executives and employees to defraud the United States".
Winterkorn faces four counts, including conspiracy to defraud the United States and wire fraud.
When the scandal broke in 2015, then-CEO Winterkorn denied having knowledge of the efforts by VW engineers to use a device and software to pass emissions inspections. He went on to say "The indictment unsealed today alleges that Volkswagen's scheme to cheat its legal requirements went all the way to the top of the company". In addition, a former manager of VW subsidiary Audi AG, an Italian citizen, has been charged but remains in Germany pending extradition.
He allegedly found out of the so-called defeat device installed in the carmaker's cars in 2014, following a study of West Virginia University's Center for Alternative Fuels, Engines, and Emissions.
"As CEO I took political responsibility", the 69-year-old Winterkorn said during a German parliamentary inquiry in January 2017, adding that, "this step was the most hard of my life".
Volkswagen admitted to cheating emissions regulations in various markets, including the United States, on a number of its diesel vehicles. VW halted the sale of new diesels in the United States after the scandal.
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