Over the last decade, an increasing number of infections with livestock-associated methicillin-resistant Staphylococcus aureus of clonal complex 398 (LA-MRSA CC398) in persons without contact to livestock has been registered in Denmark. These infections have been suggested to be the result of repeated spillover of random isolates from livestock into the community. However, other studies also found emerging sub-lineages spreading among humans. Based on genome-wide SNPs and genome-wide association studies (GWAS), we assessed the population structure and genomic content of Danish LA-MRSA CC398 isolates from healthcare-associated infections from 2014 to 2016 (n = 73) and compared these to isolates from pigs in Denmark from 2014 (n = 183). Phylogenetic analyses showed that most human isolates were closely related to and scattered among pig isolates showing that the majority of healthcare-associated infections are the result of repeated spillover from pig farms, even though cases of human-to-human transmission also were identified. GWAS revealed frequent loss of antimicrobial resistance genes and acquisition of human-specific virulence genes in the human isolates showing adaptation in response to changes in selective pressures in different host environments, which over time could lead to the emergence of LA-MRSA CC398 lineages more adapted to human colonization and transmission.