Milk is a valuable nutritious food that has a short shelf life and requires careful handling. Therefore, milk is rapidly perishable as it is a suitable medium for the growth of microorganisms particularly; bacterial pathogens that can cause milk spoilage and diseases in consumers. This 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. One hundred and thirty samples (50 raw cow’s milk, 50 raw buffalo’s milk, 10 Heat-treated milk (pasteurized), 20 Heat-treated milk (UHT)) were used for assessing the prevalence of Salmonella sp. Four Salmonella isolates were recovered from Raw cow’s milk (4/9; 8%) and five from Raw buffalo’s milk (5/9; 10%) while no isolates of salmonella sp. were detected from heat-treated milk. Moreover, the antimicrobial susceptibility testing indicated that all 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. The PCR results revealed that the six virulence genes (invA, orgA, sifA, spvA, iroN and spiA) were detected significantly in all nine isolates. Further, the results of ARDRA technique revealed that all isolates belonging to salmonella enterica and was validated using the real time- PCR. Our results indicate that insufficient hygienic measures adopted during handling. Therefore, the strict hygienic approaches are recommended during milking and processing.