Influenza A virus undergoes yearly antigenic drift that affects seasonal vaccine effectiveness. Nucleotide sequence changes primarily in the hemagglutinin (HA) gene underlie these yearly changes, and are affected by population-wide immunologic selection [1–5]. Mutation rates have been estimated to occur with a frequency of 5.72 × 10−3 nucleotide substitutions per site per year not only in the HA gene, but also independently in the matrix gene with a similar frequency of 5.39 × 10−3 nucleotide substitutions per site per year [6, 7]. Although molecular methods to detect Influenza virus RNA have targeted conserved areas of the HA and matrix genes, these methods are inherently subject to decreased sensitivity over time, as mutations accumulate in the target sequences of the assay. Examples of reduced sensitivity in molecular assays have been reported due to mutations in the Influenza matrix gene in 2012 and 2013 by Yang et al. in Taiwan , as well as in molecular assays for M. tuberculosis, and enterovirus, Respiratory Syncytial Virus, Hepatitis B, and influenza viruses . In this study, we present data suggesting that sequence differences in the Influenza matrix gene between strains of H3N2 and H1N1 account for differences in the GenMark Respiratory Viral Panel (RVP) matrix gene assay nanoamperes (nAMPs), and that these differences are reflected in the population average mean ± SDs of all positive matrix gene tests over the course of an entire season.