A newly identified subspecies of Staphylococcus hominis, S. hominis subsp. novobiosepticus, was found to be the cause of several invasive infections at a hospital in New Jersey. This subspecies differs from classical S. hominis, now S. hominis subsp. hominis, by the phenotypic characteristics of novobiocin resistance and the inability to ferment trehalose. DNA sequences of segments of 16S rRNA, DNA gyrase (gyrA), and DNA topoisomerase IV (grlA) genes were determined for the type strains of the 2 subspecies, and for 34 S. hominis clinical isolates. The 16S rRNA sequences of the type strains differed at 3 positions over 410 bp; the grlA sequences differed at 6 positions over 119 bp. These sequence differences define S. hominis subsp. novobiosepticus and S. hominis subsp. hominis "sequevars." Of 34 S. hominis clinical isolates, 31 were S. hominis subsp. novobiosepticus sequevars, 28 of which were resistant to both oxacillin and ciprofloxacin. The clinical microbiology laboratory, using a MicroScan system, identified 7 of the 31S. hominis subsp. novobiosepticus sequevars as S. hominis subsp. hominis on the basis of phenotypic characteristics. Three S. hominis subsp. hominis sequevars were all identified phenotypically as S. hominis subsp. hominis and were oxacillin- and ciprofloxacin-susceptible. Although the precise relationship between the S. hominis sequevars and their phenotypic subspecies remains to be determined, our results indicate that antibiotic-resistant clinical isolates of S. hominis belong almost exclusively to the S. hominis subsp. novobiosepticus sequevar.