The Liberal Democrats’ Jane Dodds won the mid-Wales constituency by a margin of 1,425, leaving the new Prime Minister with a working majority of just one.
This makes it far more difficult for the PM to get his policies made into law.
So what does the result mean for Boris Johnson and his government and what problems is he facing?
Will he be able to deliver Brexit?
The loss, which saw the vote swing nearly 12% from the Tories, adds to the challenges Mr Johnson faces in delivering Brexit.
The new PM was always going to face an uphill struggle to secure a Brexit deal and get it through Parliament - something his predecessor Theresa May couldn’t manage.
Even if he reaches a deal with the EU, Mr Johnson needs to drum up support from a majority of MPs to make it law.
Shaving a a piece off his already wafer-thin majority leaves him even more vulnerable to rebellions from hard Brexiteers and Remainers to block him from doing so.
The result in Brecon and Radnorshire means it will take just one Conservative MP, or a colleague from the DUP, to vote with the opposition to scupper a deal.
Mr Johnson has committed to delivering a no-deal Brexit if necessary, making a pledge that he will get the UK out of the EU on the October 31 deadline, "do or die".
He has also refused to rule out taking the controversial step of suspending Parliament in order to force his desire through against the opposition of MPs.
But there remain key figures in his own party, including David Gauke and former chancellor Philip Hammond, who will strongly oppose any no-deal attempt.
Will he call a snap general election?
Mr Johnson could call a snap general election in a bid to improve his majority in Parliament.
To do so, the Fixed-term Parliaments Act 2011 requires a motion for a snap election - which can be tabled by any party – to be supported by two thirds of the House of Commons.
It’s a gamble though - when Theresa May called an election in 2017 she ended up relinquishing seats to Labour.
The Tory leader has ruled out seeking a snap election any time soon, though that doesn’t mean he won’t make that move if he sees it as necessary.
Could he face a vote of no confidence?
Labour leader Jeremy Corbyn could try to bring down Mr Johnson with a vote of no confidence.
The move has to be backed by a majority of MPs and the latest by-election means that could be slightly more skewed towards Labour.
But it’s also a risk for Mr Corbyn, whose own party is in disarray over criticism of its Brexit stance and anti-Semitism issues.