The pair, who play Debbie Dingle and David Metcalfe in the long-running ITV soap about rural life, have a three-year-old son called Buster.
A spokeswoman for the show said: 'Charley Webb and Matthew Wolfenden have confirmed that they have separated.'
All over: Emmerdale stars Charley Webb and Matthew Wolfenden have announced they have split up
Matthew, 33, thanked fans for their kind words on his Twitter page on Wednesday after ending his five year relationship with Charley, 25.
He wrote: 'Thank you so much for all your lovely messages. Unfortunately these things happen, and it's very sad. Buster is our main priority always. X.'
The pair, who got engaged in 2009, are both expected to remain with the hit show, which celebrated 40 years on air last year.
In happier times: The couple appeared on All Star Mr & Mrs in 2012Wolfenden won reality show Dancing on Ice in 2012 and that year he said the pair had been too busy to plan their wedding.
At the time he had said: 'We don't disagree about what we want to do for our wedding. But up 'til now, we haven't had a minute to plan it.'
Webb's brother, Jamie Lomas, is married to Coronation Street star Kym Marsh and recently signed up to appear in EastEnders.
But the couple are reportedly 'living apart' and going through a trial separation while they reassess their marriage.
Neither Kim not Jamie have commented on the claims.
Staying on screen: Charley, who plays Debbie Dingle, pictured with co-star Dominic Power and Matthew both plan to stay on the show