The protesters say the minister might compromise the legal system at a time when prosecutors have to decide whether to indict Prime Minister Andrej Babis over alleged fraud involving European Union funds.
Babis denies any wrongdoing.
The justice minister has significant control over prosecutors. The protesters are angry that Marie Benesova was appointed shortly after police recommended Babis' indictment in April.
As a lawmaker, Benesova voted against a police request to strip Babis of parliamentary immunity to face investigation.
She's a lawyer and adviser to President Milos Zeman, Babis' close ally. Zeman has repeatedly criticized prosecutors.
Protesters and opposition members say she might try to influence his case, a claim that Benesova denies.
"We've had enough," and "Shame," the crowd chanted.
The fraud case involves a farm that received EU subsidies after its ownership was transferred from the Babis-owned Agrofert conglomerate of around 250 companies to Babis' family members. The subsidies were meant for medium and small businesses and Agrofert wouldn't have been eligible for them.
Later, Agrofert again took ownership of the farm.
"We are worrying about democracy," former NHL goaltender Dominik Hasek said in an address to the crowd. "We're worrying about an independent justice system."
Some of the protesters said Babis and his government should go.
"I think that the entire government should (resign)," said Eva Bernartova, a retiree who traveled from the southern town of Prachatice to join the demonstration.
"When you have the prime minister who faces criminal charges, how can he keep the government together? He's no example to follow."
After three previous protests packed Prague's Old Town Square, the demonstrations moved Tuesday to the city's much-bigger Wenceslas Square. They are set to spread across the Czech Republic next week.
The organizers also called on Babis to defend his steps in a television debate with them.
Babis, a populist billionaire, is a controversial figure because of a power-sharing deal with the Communist Party and the fraud charges. His position is also complicated by allegations he collaborated with the former communist-era secret police and accusations he's in a conflict of interests because he formally still controls his business empire whose ownership he was forced to transfer to two funds.
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