Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health. [ABSTRACT FROM AUTHOR]