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The growth in food demand and production growth of vegetables have led to the development of intensive production systems with the aim of having regular access to enough high‐quality food. The aim is to determine the incidence of Staphylococcus aureus in fresh lettuce by PCR in order to enhance the efficiency for detection and identification process. The Baird‐Parker method was used for isolating pathogens from 54 lettuce samples. Genomic DNA extraction was performed according the Mericon DNA Bacteria Plus Kit. The detection by PCR was performed using the pair of primers: coa gene (5′‐ ATAGAGCTGATGGTACAGG‐3′ and 5′‐GCTTCCGATTGTTCGATGC‐3′). The phyloge‐ netic tree was constructed by comparing conserved sequences from the adjacent 16S gene, using the F2C 5′‐AGAGTTTGATCATGGCTC‐3′ and C 5′‐ACGGGCGGTGTGTAC‐3′ primers. To test the antimicrobial effect, we used the disk diffusion method (Kirby‐Bauer) using Mueller‐Hinton agar and five antibiotics with different concentrations. The incidence of S. aureus was 1.7%. All the isolates were situated in the ATCC 11632 clade in accordance with other reported sequences belonging to this pathogen in the NCBI database. All the isolates seemed to be resistant to penicillin (10U). The molecular techniques used in this study are suitable for the identification of S. aureus isolated from lettuce, increasing our capability of detecting this pathogen by improving the process and increasing the efficiency contributing to the safety of this vegetable.

PCR Assay for Detection of Staphylococcus aureus in Fresh Lettuce (Lactuca sativa)