Milk is an excellent nutritious food that is characterized by a short shelf life and requires careful handling. Therefore, milk is rapidly perishable as it is a suitable medium for the pathogenic bacterial growth especially; causing milk spoilage and diseases in consumers. Nine Salmonella isolates recovered from 4 (8%) and 5 (10%) samples of Raw cow milk and Raw buffalo milk, respectively while no isolates of salmonella sp. were detected from heat-treated milk. Moreover, the antimicrobial susceptibility testing showed that nine isolates were resistant to Amoxicillin/clavulanic acid (AMC 30) 20/10μg, Erythromycin (E15) 15μg and Cephalexin (CL30) 30μg whilst they are all sensitive to Chloramphenicol (C30) 30μg. Additionally, our isolates revealed that they were posed 16S rRNA gene and by using amplified ribosomal DNA restriction analyses (ARDRA) technique the resulting restriction fragment patterns, showing that all isolates following the same genus and species. Importantly, two isolates were sequenced and submitted in NCBI under accession numbers OR048345 and OR102504. The virulence gene profile of the nine isolates revealed that they are poses the six virulence genes (invA, orgA, sifA, spvA, iroN and spiA) were detected significantly in all nine isolates. A mimic experiment was done to estimate the sensitivity of the real time-PCR to detect salmonella sp. in contaminated milk samples. Our results indicate that insufficient hygienic measures adopted during handling. Therefore, the strict hygienic approaches are recommended during milking and processing. The current study aimed to determine the prevalence of Salmonella sp. in raw and heat-treated milk, characterize its virulence-associated genes, and assess its antimicrobial profile.