A group of 59 putative strains of Staphylococcus intermedius/Staphylococcus pseudintermedius deposited in the Czech National Collection of Type Cultures (CNCTC, National Institute for Public Health, Prague, Czech Republic) and the National Reference Laboratory for Staphylococci (NRL for Staphylococci, National Institute for Public Health, Prague, Czech Republic) was reclassified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There the biggest human collection of S. pseudintermedius in Europe was analysed; 44 samples (75%) were of human origin. Twenty-two percent (n = 13) of the strains were isolated from animals, and two staphylococci were of unknown origin. This study revealed the prevalence of Staphylococcus pseudintermedius (94%, n = 53) vs. Staphylococcus intermedius (6%, n = 6) in the collection of human and veterinary staphylococci after reclassification. Results of PCR-RFLP analysis were verified by comparison with a repetitive element sequence-based polymerase chain reaction (Rep-PCR) analysis on 26 (44%) randomly selected strains. Due to a low-resolution ability of PCR-RFLP to separate Staphylococcus intermedius from Staphylococcus delphini, four isolates of Staphylococcus intermedius were biochemically verified further to exclude the presence of Staphylococcus delphini in the collection. Our results indicate that S. intermedius and S. pseudintermedius have occurred independently over an age-long period of their co-evolution.