Aims: The use of antimicrobial agent for treatment or growth promotion has added burden to treat infection diseases caused by pathogenic bacteria as they can acquire resistance. Salmonella is one of the major zoonotic bacterial pathogens that acts as a potential reservoir of antimicrobial resistance elements. In this study, the presence of Salmonella serotypes and the antibiogram patterns of the isolates from fecal samples of healthy cows in some selected localities in Eastern Cape, South Africa were studied. Methodology and results: Two hundred fecal samples were collected from healthy adult cows, of which 180 presumptive Salmonella isolates were recovered by conventional method. The isolates were identified using specific primer sets that are capable of detecting Salmonella spp. as well as delineating them into serogroups A, B, C1, C2, and D. Thereafter, antimicrobial susceptibility patterns of the identified isolates were determined by disk diffusion method against a panel of 12 antibiotics. From the molecular analysis of the isolates, 108 isolates were identified as Salmonella spp. and the confirmed isolates were further delineated into serogroup and the prevalence of the serogroups detected were 20%, 18%, 2%, 20% and 40% for serogroup A, B, C1, C2 and D respectively. Extremely high levels of antibiotic resistances were observed among the study isolates, while serogroup D was the most prevalent serogroup among the study isolates. Conclusion, significance and impact of study: In conclusion, dairy cows could be considered as major reservoirs of antibiotic resistant Salmonella spp. that could be transmitted to humans via the food chain. This poses a significant public health risk especially to people living around the farms as well as those who consume poorly cooked meat and those who deal on raw cow meat.