BACKGROUND: Staphylococcus aureus is a leading cause of mastitis in dairy livestock and is a pathogen with unknown but potential impact on public and herd health in Maine. The primary objective of this study was to describe retrospective trends in S. aureus detection at the University of Maine Cooperative Extension Veterinary Diagnostic Laboratory (UMVDL) for milk samples submitted between July 2017 and June 2022. The second objective was to assess the genetic profiles focused on antibiotic resistance and exotoxin genes of 29 S. aureus isolates submitted from dairy farms in Maine in 2017 and 2022. RESULTS: Overall, 7.8% of milk samples submitted to UMVDL between July 2017 and June 2022 were positive for S. aureus. The 29 isolates collected in 2017 (2 isolates) and between May and July of 2022 (27 isolates) were analyzed by whole genome sequencing and belonged to 8 strain types and 5 clonal complexes typically associated with ruminant species. Across the genomes of the 29 isolates, 14 antimicrobial resistance genes were detected, with antibiotic efflux as the primary resistance mechanism. Each isolate contained 2 to 10 staphylococcal enterotoxin genes representing 15 unique genes. lukED, lukMF', Staphylococcal superantigen-like proteins (SSLs), and hla, hlb, hld, hlgABC genes were also observed. Antimicrobial resistance and staphylococcal enterotoxin gene carriage mostly clustered with clonal complex and host species of origin. CONCLUSIONS: Whole genome sequencing identified ruminant-associated sequence types and antimicrobial susceptibility profiles consistent with other regional reports. Exotoxins with relevance to mastitis and SFP development were also identified. This study provides insight into future opportunities to study S. aureus prevalence and to survey dairy production in animal and public health contexts in Maine.