With Labour challenging in the "red wall" seat in West Yorkshire and the Liberal Democrats hoping to overturn a huge Conservative majority in Devon, defeat for the Tories will heap pressure on Mr Johnson weeks after 41 per cent of his own MPs said they did not have confidence in him.
He has suggested it would be “crazy” for him to quit if the party lost the two seats, and claimed he was “very hopeful” about the results.
Mr Johnson will be monitoring the results from Rwanda, where he is attending the Commonwealth nations' leaders summit.
“I’m going to be watching the results with interest but always full of optimism and buoyancy," he said in Kigali.
"But most seasoned political observers know that by-elections in mid-term are never necessarily easy for any government.”
Despite Mr Johnson's claims, a senior Tory source acknowledged that the situation was looking “extremely difficult” in the Devon seat, where the Conservatives were defending a majority of more than 24,000.
In Wakefield, a Labour campaign source said Sir Keir Starmer’s party would “just” win the contest to replace Imran Ahmad Khan, who quit after being found guilty of sexually assaulting a 15-year-old boy – a crime for which he was jailed for 18 months.
Wakefield was one of the red wall seats won by the Tories in the 2019 general election after being Labour since the 1930s.
Wakefield by-election - in pictures
In Tiverton and Honiton, Neil Parish, the Tory MP since 2010, resigned after admitting he had watched pornography on his phone in the House of Commons.
The Liberal Democrats hope to recreate by-election wins in North Shropshire in December and Chesham and Amersham a year ago by taking a Tory heartland seat.
Lib Dem MP Christine Jardine said the party’s candidate Richard Foord had “stood up for his local community” against “a Conservative party which has taken people in Devon for granted”.
“Never in the history of British politics has a majority this big been overturned in a by-election. We’ve said throughout this campaign this is a huge mountain to climb,” Ms Jardine said.
“However, if the Conservative Party lose significant numbers of votes tonight in one of their safest seats in the country, there will be countless Conservative MPs looking over their shoulders nervously tomorrow.”
What do voters have to say in UK Tiverton and Honiton by-election? Video
Mr Johnson urged voters in the rural seat to “stick up for British food and farming” by supporting Tory Helen Hurford, a former head teacher.
Losing the Tory stronghold would be regarded as a sign of Mr Johnson’s diminishing electoral appeal after scandals around lockdown parties at Downing Street and amid the cost-of-living crisis.
It could also spark more backlash against Mr Johnson's authority.
The Tories face a steeper challenge in keeping the Wakefield seat, with Labour odds-on favourite to overturn the 2019 Conservative majority of 3,358.
Tory candidate Nadeem Ahmed raised eyebrows by arguing last week that voters should still trust the party after Mr Khan’s sexual assault conviction, just as they still trust GPs despite the crimes of mass murderer Harold Shipman.
Sir Keir has said a victory for candidate Simon Lightwood in the northern constituency “could be the birthplace of the next Labour government”.
If the Conservatives were to lose both by-elections, it would be only the seventh time a government has suffered a double defeat since the Second World War.
“Labour has run positive campaigns with fantastic candidates in both by-elections," said Shabana Mahmood, the party’s national campaign co-ordinator.
"But we know that taking the seats requires many Tory voters to switch to Labour, which is harder in by-elections where low turnout is commonplace.
“Wakefield has been a marginal constituency since Labour last won a general election, and the Conservatives hold the seat with the biggest majority since 2010, making this an uphill task.”