She forgave 52-year-old Montgomerie after the ex-Ryder Cup captain embarked on a secret relationship with an ex-girlfriend, Joanne Baldwin, only two years into their marriage.
He said at the time: 'I have put my marriage under considerable strain but we are working through these problems. I am very sorry for the hurt I have caused to the ones I love so much.'
And when his relationship was back on track, he expressed his happiness, saying: 'I'm thrilled to say we've worked at it and everything is good now.'
But the pair have now parted ways, with Montgomerie said to be dividing his time between staying in hotels and at his father's home in Ayrshire.
Mrs Knowles is now said to be 'furious' and preparing divorce papers.
A source said: 'Colin decided to call it a day and Gaynor was devastated. She is furious after taking him back and getting around to filing divorce papers. She wants it done now.'
Montgomerie split up with his first wife Eimear Wilson, his childhood sweetheart, in 2004 after 14 years of marriage and three children together. It was reported she had given him an ultimatum - golf or their marriage.
After marrying Mrs Knowles in 2008, it emerged in 2010 he had rekindled his friendship with Miss Baldwin, a 50-year-old mother-of-two, following a relationship they had struck up in 2005.
The passionate affair lasted for several months before Montgomerie confessed to his wife.
In the early days of his relationship with Mrs Knowles, he blew £30,000 on a private jet to take her from Germany - where he was competing in a golf tournament - to Glasgow to watch a Robbie Williams concert.
The Scottish golfer then flew back to Munich in time to tee off the next day at the BMW international open - a roundtrip of 2,000 miles.
Montgomerie is believed to have been introduced to Mrs Knowles, a multimillionaire widow, by Scots BBC sports presenter Dougie Donnelly.
Montgomerie has previously been linked to Spanish model Ines Sastre, former Miss England Helen Westlake and BBC journalist Alison Walker.
He was spotted at Wimbledon with catwalk model Alex Leigh, who is 16 years his junior. The pair denied any romance.
In a statement released last night, Montgomerie said: 'I can confirm that I raised proceedings some time ago seeking divorce from Gaynor.
'We are separated and in the process of divorcing. There are no third parties involved and I ask that you respect our privacy at this difficult time.'