The departure of Suella Braverman, over a "technical" breach of government rules, means Truss has lost two of her most senior ministers in less than a week, both replaced by politicians who had not backed her for the leadership.
Hours after the resignation on Wednesday, MPs openly rowed and jostled amid confusion over whether a vote on fracking was a confidence vote in her administration.
Opposition parliamentarians complained that Truss's politicians were being manhandled to make them vote with the government, though two MPs from her Conservative party said they had not seen any such behaviour.
"Discipline is falling apart, we can't go on like this," one Conservative MP told Reuters.
Another, Charles Walker, told BBC television he was "livid" at the "talentless people" who had put Truss into power, just because they wanted a job.
"I think it is a shambles and a disgrace," he said.
Truss, in power for just over six weeks, has been fighting for her political survival since September 23, when she launched a mini-budget of vast unfunded tax cuts that sent shock waves through financial markets.
A handful of MPs have openly called for her to quit, and others have discussed who should replace her.
Following the scenes in parliament, there were reports the Conservative chief whip and her deputy had quit, but Truss's office said they remained in their posts.
The latest drama at Westminster comes as millions of Britons worry about rising inflation and cuts to public spending, with the political elite seen to be doing little to ease those concerns.
Truss had just told MPs she was sorry for her mistakes but was not going to quit, when reports swirled that her interior minister, or home secretary, had gone.
Confirming her departure, Braverman said she had broken the rules by sending an official document from her personal email to a parliamentary colleague.
But she said she had serious concerns about the government and that just hoping problems would go away was not a viable approach.
"I have made a mistake, I accept responsibility, I resign," she said in a letter to the prime minister.
As a replacement, Truss appointed former minister Grant Shapps, who said recently that Truss faced a massive battle to survive.
Media reports suggested Truss and Braverman might have clashed over immigration.
Braverman, who said recently that she dreamt of seeing asylum seekers being deported to Rwanda, has advocated a hard line on immigration numbers, while Truss has suggested restrictions could be lifted in some sections of the economy.
After a move to make a vote on fracking a confidence matter, scenes in parliament descended into chaos as Conservative MPs were confused over whether they were obliged to vote in line with the government.
The motion by the opposition Labour Party was defeated by 326 votes to 230 and the government proposal won, but some MPs said they were angry over the government's tactics, or lack of them.
Hours earlier, Truss had faced a raucous questions session for the first time since new chancellor Jeremy Hunt scrapped most of her tax-cutting plan.
Asked by Labour leader Keir Starmer why she should remain in power, she said: "I am a fighter and not a quitter."
"I have been very clear that I am sorry, and I have made mistakes," Truss told jeering opposition MPs in parliament. "I'm prepared to take the tough decisions."
The Conservative Party is some 30 points behind Labour, according to opinion polls, and YouGov ranks Truss as the most unpopular leader the pollster has ever tracked.
