Matuidi joined Inter Miami on a free transfer in August after leaving Juventus.
The 33-year-old France world cup winner was signed with Transfer Allocated Money rather as a designated player, which would have seen him earn a higher salary.
Nonetheless the MLS authorities believe Matuidi is being paid more than his contract stipulates and should have been registered as a designated player.
A statement read: "Major League Soccer has begun a formal review of Inter Miami CF’s signing of midfielder Blaise Matuidi, specifically investigating whether the signing of the player complied with Major League Soccer’s salary budget and roster guidelines.
"MLS will not make further comment until the completion of its investigation."
Miami, co-owned by former England captain, David Beckham, have acknowledged receipt of the investigation and are willing to comply.
The club has welcomed the investigation, saying in a statement: "Inter Miami CF acknowledges the statement made by Major League Soccer today, and we look forward to fully engaging with the league's review process.
"We will have no further comment."
Matuidi made 16 appearances and scored one goal for Inter Miami last season.
