The FBI has obtained a warrant to start reviewing emails found on a laptop used by top Hillary Clinton aide Huma Abedin and her husband Anthony Weiner, according to reports from US media.
The Wall Street Journal says the number of emails could total 650,000 – though not all of them are expected to be relevant to the Clinton investigation.
In a letter to Congress, the agency’s director, James Comey, said new information had prompted an additional review of the case that ended in July.
Former Congressman Mr Weiner is being investigated separately after claims he exchanged illicit text messages with a teenager.
The FBI conducted a months-long investigation into whether Mrs Clinton had broken the law by using a private email server during her time as US Secretary of State, by allowing sensitive information to be discussed outside a secure government account.
The FBI concluded that while Mrs Clinton had been “extremely careless” she had not committed a crime.
Mr Comey’s disclosure has sent shockwaves through the final days of the presidential race.
Democrats have come out fighting, insisting Mr Comey should have investigated further before making the announcement so close to the 8 November election.
Senate Democratic leader Harry Reid says Comey may have broken the law by making the disclosure at this stage of the campaign.
He claims the FBI director appears to be aiding one political party over another – a move that may violate the Hatch Act, which bars government officials from using their position to influence an election.
Donald Trump, however, is seizing a moment that puts his opponent in the spotlight.
He told a rally in New Mexico on Sunday that the FBI had found the “mother lode”.
“How do you have that many emails? What do you do, sit down all day and just keep typing?” he asked.
“No wonder nothing gets done in our country.”
He added: “This is the single biggest scandal since Watergate.”
The Republican has been on the back foot for weeks following a hot mic recording of him boasting about sexual assault which prompted 12 women to come forward and accuse him of groping or kissing them without consent.
Earlier, he told an energised crowd in Las Vegas that the only way to put a check on Mrs Clinton’s corruption was to use the “power of the vote”.
A new Washington Post-ABC poll found that just over three in 10 voters said the news made them less likely to support Mrs Clinton. About two thirds of those voters were Republican or Republican leaning.
More than six in 10 likely voters said the FBI’s announcement would make no difference in their vote.
The true impact of this latest revelation may not be truly reflected until the middle of this week.