Influenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. Here, we use deep mutational scanning to measure the effect of every amino-acid substitution in M1 on viral replication in cell culture. The map of amino-acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell-culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCE The M1 matrix protein is critical for many stages of the influenza infection cycle. Currently, we have an incomplete understanding of this highly conserved protein´s function and structure. Key regions of M1, particularly in the C-terminus of the protein, remain poorly characterized. Here we used deep mutational scanning to determine the extent of M1´s tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1, and shape future inquiries probing the function of this essential influenza A virus protein.