Members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5–100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.