The present study was carried out in an attempt to detect the distribution of antibiotic-resistant genes of Staphylococcus aureus isolates from human infections and bovine mastitis. mecA, msrA, msrB, aacA-D, tetK and tetM genes were selected in order to detect the distribution of antibiotic-resistant genes by multiplex PCR technique. According to the biochemical analysis and detection of 16S-rDNA by PCR method, 108 isolates of 300 human infections samples and 18 strains from 150 bovine mastitis milks were recognized as S. aureus. Distributions of antibiotic-resistant genes in human isolates were as follows: (85.18%) mecA, (46.29%) msrA, (49.07%) msrB, (33.33%) aacA-D, (80.50%) tetK and (66.66%) tetM and in bovine mastitis, isolates were seen to be ranging from: (22.22%) mecA, (66.66%) msrA, (77.77%) msrB, (33.33%) aacA-D, (55.55%) tetK and (50.00%) tetM, respectively. Results indicated that all S. aureus strains have one or more of the antibiotic-resistant genes. Also, multiplex PCR technique is a fast, practical and appropriate technique for determining antibiotic-resistant genes. Hence, it was possible that the treatment and the right antibiotics were used.