Species identification of 200 beta-hemolysin-producing canine staphylococcal isolates was performed using a recently described polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method (based on MboI restriction of a pta gene fragment), supplemented with biochemical testing and sequencing of housekeeping genes. The PCR-RFLP method misclassified a small fraction (approximately 1%) of the Staphylococcus pseudintermedius population as a result of heterogeneity in the MboI restriction site. A potentially novel species within the Staphylococcus intermedius group (SIG) was found, having closest similarity to S. intermedius based on sequence comparison to the genes sodA, pta, hsp60, tuf, and full-length 16S ribosomal DNA, thus demonstrating further species diversity within the SIG.