Thomas, a two-time major champion and 15-time PGA Tour winner, had hired Mackay in 2021 following the latter's lengthy run on the bag with Phil Mickelson.
"While incredibly difficult for me to say, Bones and I have parted ways," Thomas wrote on social media.
"I'm going to be forever thankful for him joining me on the bag in 2021.
"The things we've been able to accomplish together -- the PGA Championship in 2022, the Presidents Cup, the Ryder Cups were all unforgettable experiences.
"His wisdom on and off the course has been a blessing during a tough stretch of my career and he was there every step of the way.
"I know there are great things coming for both of us down the road. I wish him the best of luck and will always count him and his family amongst my friends."
Thomas, 30, is currently 28th in the Official World Golf Ranking. He did not divulge who will be his caddie at Augusta National Golf Club next week.
He's started the season well, finishing among the top 12 in his first three events, including a tie for third at the American Express in La Quinta.
Thomas then failed to make the cut at the Genesis Invitational, tied for 12th at the Arnold Palmer Invitational, missed the cut again at The Players Championship and finished tied for 64th at Valspar Championship.
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