Mastitis is considered the most predominant production infectious disease in dairy industry all over the world including Egypt, The difficulty of mastitis occurs due to its chronic nature of the disease, extremely economic losses, extensively spread all over the year and intense decrease in the milk production. Staphylococcus aureus is one of the most common bacterial agents causing mastitis and having public health importance. A total of 248 milk samples were collected from different dairy farms, 77 samples from mastitic milk, 171 from apparently normal milk. 24 samples were positive for S. aureus by applying coagulase test. This study is concerned with the detection of thermonuclease (NUC gene) of S. aureus through PCR reaction, sequencing and phylogenetic analysis. Sequence alignments and phylogenetic analysis revealed high level of similarities between the different alleles isolated from different samples. Genotyping and sequencing analysis of NUC gene is a good tool to understand the relationships and differences between different isolates of S. aureus causing mastitis in dairy farms.