Some Staphylococcus species are believed to be the main cause of bacterial infections and foodborne outbreaks. Several reports have discussed the enterotoxigenic properties of some Staphylococcus species, but due to the shortage of efficient diagnostic techniques, most studies have focused only on Staphylococcus aureus. Thus, developing a culture-independent, selective, and rapid detection method for Staphylococcus species in food products is of great importance. In this study, PCR-amplified tuf gene sequences were assessed by temporal temperature gradient gel electrophoresis (TTGE) in order to detect and differentiate between different Staphylococcus species in Iranian food samples. The PCR sensitivity and specificity were evaluated against DNA samples extracted from six Staphylococcus species, including S. aureus, S. epidermidis, S. saprophyticus, S. intermedius, S. chromogenes, and S. hominis, using a commercially available kit and a cost-effective, rapid, non-commercial boiling method. Using the boiling method, the sensitivity of the tuf PCR was 9 × 101 CFU/mL for the salami samples spiked with S. aureus, ten times less sensitive than the commercial kit. After optimizing the TTGE conditions, a species-specific TTGE pattern was obtained based on the differences between the amplified sequences from various species. This TTGE pattern was applied to detect Staphylococcus species in food samples from the market. The presence of Staphylococcus species was confirmed in 6 out of 10 tested salami products. The results demonstrate that the PCR-TTGE method is an alternative method that may be specific and sensitive enough to assess the presence of possible Staphylococcus contamination in meat processed food samples. More studies using different food samples should be considered for an in-depth analysis of bacterial contamination.