Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be nonpersistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type tbl 2645 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type tbl 2645 were less likely to cause a persistent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than tbl 2645 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada.