We evaluated the cell invasion ability (CIA) of non-invasive Streptococcus dysgalactiae subsp. equisimilis using human keratinocytes and determined the association of CIA populations with their hosts and microbiological traits. Forty-two isolates from humans and companion animals were selected with host information. In addition to CIA, virulence-associated gene (VAG, spegg–ska–scpA–inlA–sicG–brpA–prtF1–prtF2–lmb–cbp–srtp1–srtp2) profiling, emm genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping were performed. We designated CIA values higher than the mean of all isolates as high-frequency and those lower than the mean as low-frequency. Differences in the CIA between the different sources and Lancefield groups were assessed. We analyzed the association between high- and low-frequency CIA and VAG, emm genotype, sequence type/clonal complex, and AMR phenotype/genotype. Based on the mean (19.368 colony-forming units/100 cells) of 42 isolates, eight isolates had high-frequency CIA, whereas 34 had low-frequency CIA. We found an association between low-frequency CIA population and group G isolates, as well as a link between high-frequency CIA population and group C isolates. We also observed associations between low-frequency CIA population and oral/respiratory tract origin, ska, scpA, and lmb detection, and the AMR phenotype. Our observations suggest potential associations between high-/low-frequency CIA and the group, source, VAG, and AMR phenotypes.