| Summary: | <i>Salmonella enterica</i> is a leading cause of human gastrointestinal disease worldwide. Given that <i>Salmonella</i> is persistent in aquatic environments, this study examined the prevalence, levels and genotypic diversity of <i>Salmonella</i> isolates recovered from major rivers in an important agricultural region in northwestern Mexico. During a 13-month period, a total of 143 river water samples were collected and subjected to size-exclusion ultrafiltration, followed by enrichment, and selective media for <i>Salmonella</i> isolation and quantitation. The recovered <i>Salmonella</i> isolates were examined by next-generation sequencing for genome characterization. <i>Salmonella</i> prevalence in river water was lower in the winter months (0.65 MPN/100 mL) and significantly higher in the summer months (13.98 MPN/100 mL), and a Poisson regression model indicated a negative effect of pH and salinity and a positive effect of river water temperature (<i>p</i> = 0.00) on <i>Salmonella</i> levels. Molecular subtyping revealed Oranienburg, Anatum and Saintpaul were the most predominant <i>Salmonella</i> serovars. Single nucleotide polymorphism (SNP)-based phylogeny revealed that the detected 27 distinct serovars from river water clustered in two major clades. Multiple nonsynonymous SNPs were detected in <i>stiA</i>, <i>sivH,</i> and <i>ratA</i>, genes required for <i>Salmonella</i> fitness and survival, and these findings identified relevant markers to potentially develop improved methods for characterizing this pathogen.
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