By Alexander Osipovich

Mystery traders used advance knowledge of a Chinese brokerage crackdown to make more than $100 million in profit from well-timed options trades, one of the world's biggest electronic-trading firms alleged in a lawsuit.

Two units of Susquehanna International Group filed the lawsuit in federal court in New York on Monday, naming up to 100 "John Does" as the defendants.

Susquehanna — which said it took a big loss after being caught on the wrong side of the trades — is seeking to uncover the traders' identities and force platforms that facilitated the transactions to freeze their accounts.

The timing and nature of the options bets strongly suggested that the unknown traders knew in advance that Chinese regulators were moving to penalize Futu and UP Fintech, the parent company of Tiger Brokers, Susquehanna said.

Futu and Tiger Brokers are online brokerages popular in Hong Kong and other Asian markets. On May 22, the China Securities Regulatory Commission said that it had found them to be illegally serving customers in mainland China without the proper licenses, a move that triggered a sharp drop in the share price of the two companies.

Both Futu and Up Fintech have U.S. shares listed on the Nasdaq. Susquehanna alleged that the mystery traders cashed in on their inside information by buying large quantities of bearish put options that would pay out if the price of Futu and UP Fintech stock dropped.

The traders bought the options contracts for about $12 million and earned a profit of more than $100 million, reflecting a return of more than 900%, Susquehanna said.

The profit makes the alleged scheme "one of the largest documented cases of insider trading in recent memory," Susquehanna said in the lawsuit.

By comparison, the total illegal profit from the insider-trading scheme masterminded by hedge-fund titan Raj Rajaratnam was $53.8 million. The founder of hedge fund Galleon Group was convicted of securities fraud and conspiracy in 2011.

Susquehanna itself lost $71.4 million by being caught on the wrong side of the options trades, it said in the lawsuit, offering a rare glimpse of a trading loss at the secretive options powerhouse.

The Pennsylvania-based firm led by billionaire Jeff Yass is one of the largest market makers in U.S. options, meaning that when investors buy or sell options, Susquehanna often takes the opposite side of the bet.

Known for its use of poker to train young traders, Susquehanna uses sophisticated quantitative models to determine what price it is willing to buy or sell at, ensuring that it makes money as it trades vast quantities of stocks, options and futures every day.

But in the case of the Futu and UP Fintech trades, Susquehanna said it faced an even more knowledgeable opponent — traders who knew the companies were in the crosshairs of regulators in Beijing.

From May 7 to 21, there was a significant increase in purchases of put options on Futu and UP Fintech, according to Susquehanna's lawsuit. Many of those contracts would only pay out if the stocks dropped sharply, well below their prices at the time that the mystery traders bought the options. Many were also "short-dated," meaning that if there hadn't been a big price drop within the coming days, the contracts would have expired worthless.

The trades "thus suggest inside knowledge not only of the fact that the crackdown news was going to be announced, but also its approximate timing," Susquehanna said.

Susquehanna's lawsuit said that two possible categories of John Does were behind the trades: regulators at the CSRC or personnel at Futu or UP Fintech who knew the crackdown was imminent.

The CSRC, Futu and UP Fintech didn't respond to requests for comment. Immediately after the May 22 crackdown, both brokerage firms said they were cooperating with the Chinese regulator.

Most of the trades were carried out by about nine accounts at Interactive Brokers, a U.S.-based brokerage used by clients around the world, while a smaller number of the trades were executed though platforms owned by Futu or UP Fintech, according to the lawsuit.

Interactive Brokers said in a statement: "We have been cooperating with Susquehanna, including freezing accounts. We will continue to cooperate fully with all regulatory investigations and court proceedings."

Write to Alexander Osipovich at alexo@wsj.com