Nominations for the top job closed on Friday ahead of the group's annual general meeting on May 14.
The decision of his colleagues not to stand against him means Mr Matheson, who represents Anderston/City, will lead the city council into the Commonwealth Games.
He said: "I am proud and humbled to have again been elected unopposed as leader of the Labour Group at Glasgow City Council.
"As I start my fifth year as city leader, there is a real buzz about Glasgow and I and my Labour colleagues will build on our successes over the coming year.
"2014 is the year of Glasgow. We will be on the world stage like never before when we host the Commonwealth Games and the MTV European Music Awards. We will capitalise on all the opportunities this year provides.
"Looking beyond 2014, our negotiations with the UK Government for a City Deal, and our work with Sustainable Glasgow and Future Cities is helping to shape Glasgow for a smart, low-carbon and metropolitan future."
Mr Matheson, who educated at Glasgow and Strathclyde universities, was first elected to the city council in 1999.
He was the councillor responsible for education and became city treasurer before being elected leader in May 2010.
In the May 2012 local government elections he led the Scottish Labour Party to victory, increasing the share of the vote by 3.4% and securing an overall majority in Glasgow.
That was despite predictions from many political pundits that Labour would lose control of Glasgow to the SNP.