In recent years, coagulase-negative staphylococci (CoNS) have been increasingly recognised as causative agents of various infections, especially in immunocompromised patients and related to implanted foreign body materials. In this study, rpoB sequencing was used for simultaneous species identification and detection of rifampicin resistance in clinical staphylococci isolates. Forty-nine (96%) out of 51 isolates, representing 17 different Staphylococcus species according to the initial phenotypic species identification, were identified to the species level using rpoB sequencing. Furthermore, the two remaining isolates were Kocuria sp. and Corynebacterium sp. respectively, according to 16S rRNA sequencing. Comparison with the phenotypic diagnostics also revealed that 8 (16%) of the 49 isolates differed regarding identified species. Discrepant analysis confirmed the result of the rpoB sequencing for all except 2 of these isolates, which could not be distinguished as single species using 16S rRNA sequencing. Regarding detection of rifampicin resistance, isolates obtained pre- and post-treatment with rifampicin were examined. These isolates comprised S. aureus (7 patients) and S. lugdunensis (1 patient). Rifampicin resistance was mainly detected following short-term treatment with rifampicin in combination with isoxazolyl-penicillin, or long-term treatment with rifampicin and ciprofloxacin. Each rifampicin-resistant isolate displayed an identical rpoB sequence as their corresponding rifampicin-susceptible isolates except for one (n = 6) or two (n = 1) nonsynonymous single nucleotide polymorphisms, or insertion of one codon (n = 1). In conclusion, rpoB sequencing is a rapid, objective and accurate method of species identification and simultaneous detection of rifampicin resistance in staphylococci.