Equine influenza virus (EIV) is a major infectious pathogen causing significant respiratory signs in equids worldwide. Voluntary surveillances in the United States (US) recently reported EIV detection in horses with respiratory signs even with adequate vaccine protocols and biosecurity programs and posed a concern about suboptimal effectiveness of EIV vaccine in the US. This study aims to determine genetic characteristics of 58 field EIV H3N8 strains in the US from 2012 to 2017 using the phylogenetic analysis based on hemagglutinin (HA) gene. Amino acid substitution and acquisition of N-glycosylation of the HA gene were also evaluated. Phylogenetic analysis identified that almost all US field strains belonged to the Florida Clade 1 (FC1) except one Florida Clade 2 strain from a horse imported in 2014. US EIV strains in 2017 shared 11 fixed amino acid substitutions in the HA gene comparing to the vaccine strain (A/equine/Ohio/2003) and two additional amino acid substitutions were detected in 2019. Introduction of foreign EIV strains into the US was not detected, but antigenic drift without acquisition of N-glycosylation in the HA gene were observed in US field strains until 2017. Considering the global dominance of FC1 strains, subsequent antigenic drift of US EIV strains should be monitored for better effectiveness of EIV vaccine in the US and global equine industries