The “Flixtrains”, decked out in the trademark bright-green of the company's low-cost buses, will travel from Hamburg to Cologne from March 24th, while the Stuttgart to Berlin line will open in April.
Prices will start from €9.99, lower than those of rival Deutsche Bahn.
“We already proved with Flixbus that mobility doesn't have to be expensive,” company founder Andre Schwaemmlein said in a statement.
“With the integration of Flixtrain, the options for German travellers are becoming even more attractive.”
Germany's rail sector was liberalised in 1994 but remains dominated by Deutsche Bahn, which still accounts for 99 percent of all long-distance rail journeys.
The Flixbus company will not own its Flixtrains, partnering instead with Czech rail operator Leo Express and Nuremberg operator BahnTouristikExpress.
The firm first dipped its toes in the rail business last year when it came to the rescue of insolvent start-up Locomore, allowing it to resume its low-cost Berlin to Stuttgart route.
Founded just five years ago, Flixbus has grown into Germany's most popular long-distance bus company and has since expanded into 25 other European countries, transporting over 100 million people.
The low-cost giant has now set its sights on the United States, where it plans to take on the iconic Greyhound Lines by launching a series of long-distance bus services in California later this year.