Streptococcus uberis isone ofthe most important mastitis-causing pathogens. Although the pathogenesis and virulence factors required for the intramammary infection development are not yet well established, several putative virulence-associated genes have been described. This work aimed toinvestigate the presence often known and putative virulence-associated genes inS.uberis isolated from subclinical orclinical mastitis and its closely related species Streptococcusparauberis in135dairy farms inthe Czech Republic. The PCR analysis detected that all the examined isolates possessed atleast four virulence genes and most isolates carried eight out often virulence genes. All S.uberis isolates were positive for the oppF, gapC and sua genes. Among the most prevalentvirulence-associated genes skc (98%) and pauA (97%) were also found. The hasA and hasB genes were always present together in94% ofthe isolates. The genes cfu and lbp were detected in6% and 2%, respectively. IntheS.uberis isolates, 14different virulence gene profiles were observed. The most frequent profile was hasA + hasB + sua + skc + pauA + gapC + oppF with variable hasC, observed in86% ofthe tested isolates, occurring in127out of135farms. S.parauberis was identified very sporadically and, although itis closely related toS.uberis, only arare occurrence ofthe examined virulence-associated genes was found.