Matthews is not feeling any symptoms and the team is waiting for the results of his PCR test to confirm if he has COVID-19.
Assistant coach Dean Chynoweth was also held out of practice after he also tested positive for COVID-19 on his rapid test.
Toronto has been working through a COVID-19 outbreak that saw 14 players and seven staff members placed in the NHL's coronavirus protocol at its height. Leafs defenceman Timothy Liljegren was the only player still in isolation when they beat the visiting Ottawa Senators 6-0 on Saturday, but he returned on Monday.
Matthews is coming off an outstanding December that saw him named the NHL's first star of the month after putting up 10 goals and four assists in seven games.
The 24-year-old tested positive for COVID-19 in June 2020 at home in Arizona while the 2019-20 season was suspended due to the pandemic.
The Leafs are set to host the Edmonton Oilers on Wednesday. Toronto's home game scheduled for Monday night against the Carolina Hurricanes was one of a number of contests postponed by the NHL due to arena capacity restrictions in Canada.
- With files from the Canadian Press.