Salmonellosis is a major cause of gastrointestinal infection that generally occurs through the ingestion of fecally contaminated food or water. Molecular markers based on the gyrase B (gyrB) gene sequence, a Type II DNA topoisomerase subunit enzyme family member, were developed to specifically and sensitively discriminate Salmonella spp. from closely related and collocated microorganisms in a water environment. For this, gyrB gene sequences of Salmonella spp., E. coli, Yersinia enterocolitica, Klebsiella pneumoniae and Shigella spp. were aligned and found up to 88% similar. Markers amplified from primers specific to the Salmonella gyrB sequences were conserved across 13 S. enterica and one S. bongori serovar, and were able to detect approximately one S. Enteritidis genome. These were more specific and sensitive than the international standard invA gene-based Salmonella marker. The gyrB markers detected 725 Salmonella genomes in 100 mL of seeded environmental water sediment sample and 72 Salmonella genomes in 300 g of seeded minced samples. Successful detection of Salmonella in non-inoculated minced samples was also achieved, with higher sensitivity than the invA markers. These makers should be useful in future risk analyses and standards setting for Salmonella presence in food, and water used for irrigation and recreational purposes.