The duo have won numerous tournaments, including this year's US Open and a silver medal at the 2012 Olympic Games in London.
However, the decision to end the partnership was taken by Hlavackova, who has decided to move her career in another direction.
"It was Andrea's decision. She told me she wanted to play with Lucie Safarova next year and focus on singles," 28-year-old Hradecka told Tennis Arena.
Meanwhile, Hlavackova added: "I would like to thank Lucie Hradecka for those five years together and a lot of tournaments that we had taken place.
"It was an amazing part of my career. Of course I wish her good luck in the coming years."
The pair could still play alongside each other in the Fed Cup.