Three hundred and sixty different samples were collected from different sources, including wound, burn, nasal, and oral swabs from several hospitals in Baghdad. A number of 150 (53%) Staphylococcus aureus samples were isolated and identified among a total of 283 samples. Then, the spread of the Toxic Shock Syndrome Toxin-1 gene (tsst-1) was investigated in β-lactamase resistant S. aureus. According to the source of samples, the distribution of S. aureus  isolates was found to be significantly higher (p < 0.01) in wound samples as compared to other sources. According to the age, a highly significant  distribution (p < 0.01) was recorded in the age group of 15-30 years, whereas gender comparison showed no statistically significant differences. All the isolates were subjected to susceptibility test against eight β-Lactam antibiotics by using the disc diffusion method. The antimicrobial susceptibility test showed that S. aureus had maximum resistance percentage to Carbenicillin (97.3%), while the lowest resistance rate was against Meropenem, with a sensitivity rate of up to 82%. In addition, 144 (96%) out of the 150 S. aureus isolates have multiple drug resistance (MDR). All the isolates were subjected to polymerase chain reaction to amplify tsst-1 toxin gene. A number of 70 isolates (46.7%) were found to be positive for tsst-1 gene. The results showed no significant correlation between tsst-1 gene with the individual antibiotic resistance and the multi-drug resistance patterns of the isolates (p = 0.226).