Matheson repaid the bill in full and apologized. However, Matheson said he did not want the debate about the bill to become a distraction and resigned.
His resignation comes ahead of the publication of an investigation into the incident by the Scottish Parliament's Corporate Body, which is examining whether Matheson made an inappropriate expense claim.
In his resignation letter to Prime Minister Hamza Yusuf, Matheson said he had still not received the body's report.
Matheson said in his letter:
"However, to ensure that this situation does not become a distraction from advancing the government's agenda, it is in both my and the government's best interest that I resign."
"I agree that it is best for you to resign now to ensure that you can give the parliamentary process the attention it deserves without becoming a distraction to advance the government's agenda," Yusuf said, regretting the resignation.
Neil Gray has replaced Matheson as Minister of Health.
