The aim of this study was to apply the Restriction Fragment Length Polymorphism (RFLP) of Glyceraldehyde-3-Phosphate Dehydrogenase encoding gene (gap) for testing its performance as a molecular typing tool in coagulase negative staphylococci (CNS) isolates from bovine mastitis (n = 59) and human clinical cases (n = 13) identified with VITEK 2. According to the phenotypic identification results, bovine mastitis isolates were Staphylococcus haemolyticus, Staphylococcus simulans, Staphylococcus auricularis, Staphylococcus warneri, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus xylosus, Staphylococcus epidermidis and Staphylococcus cohnii. Although most of those isolates were generated PCR amplicons with gap gene specific primers, PCR amplification of gap gene failed in 29 from 72 isolates. The samples that did not produce amplicons were reamplified with Staphylococcal 16S rRNA gene specific primers. After PCR amplifications, amplicons were produced in 17 from 29 samples. Three different restriction endonucleases (AluI, MseI and RsaI) were used for PCR-RFLP analysis, among these AluI has been found the most discriminatory power for identification in species. The results of the RFLP of gap gene provide a support for the misidentification problem associated with VITEK 2 system for S. simulans, S. auricularis and S. capitis species. Moreover, more frequent failure in gap gene amplification for bovine isolates which were phenotypically identified as S. simulans, S. auricularis, S. capitis, S. xylosus and S. cohnii was not clear. In addition, the method verified the phenotypic identification for S. haemolyticus, S. warneri, S. hominis and S. epidermidis isolates with different rates at 100, 33.3, 57.1, and 66.7%, respectively.