This study examined the diversity and persistence of Salmonella in the surface waters of agricultural regions of Brazil, Chile, and Mexico. Research groups (three in 2019-2020 and five in 2021-2022) conducted a long-term survey of surface water across 5-8 months annually (n = 30 monthly). On-site, each team filtered 10-L water samples with modified Moore Swabs to capture Salmonella, which were then isolated and identified using conventional microbiological techniques. Salmonella isolates were sequenced on Illumina platforms. Salmonella was present in 1,493/3,291 water samples (45.8%), with varying isolation rates across countries and years. Newport, Infantis, and Typhimurium were the most frequent among the 128 different serovars. Notably, 22 serovars were found in all three countries, representing almost half of the 1,911 different isolates collected. The resistome comprised 72 antimicrobial resistance (AMR) genes and six point mutations in three genes. At least one AMR determinant was observed in 33.8% (646/1,911) of the isolates, of which 47.4% (306/646) were potentially multidrug resistant. Phylogeny based on core genome multilocus sequence typing (cgMLST) showed that most isolates clustered according to sequence type and country of origin. Only 14 cgMLST multi-country clusters were detected among the 275 clusters. However, further analysis confirmed that close genetic relatedness occurred mostly among isolates from the same country, with three exceptions. Interestingly, isolates closely related phylogenetically were recovered over multiple years within the same country, indicating the persistence of certain Salmonella in those areas. In conclusion, surface waters in these regions are consistently contaminated with diverse Salmonella, including strains that persist over time.IMPORTANCESalmonella is a leading foodborne pathogen responsible for millions of illnesses, hospitalizations, and deaths annually. Although Salmonella-contaminated water has now been recognized as an important contamination source in the agrifood chain, there is a lack of knowledge on the global occurrence and diversity of Salmonella in surface water. Moreover, there has been insufficient research on Salmonella in surface waters from Latin American countries that are major producers and exporters of agricultural products. Incorporating genetic profiling of Salmonella isolates from underrepresented regions, such as Latin America, enhances our understanding of the pathogen's ecology, evolution, antimicrobial resistance, and pathogenicity. Moreover, leveraging genomic data derived from pathogens isolated from diverse geographical areas is critical for assessing the potential public health risk posed by the pathogen and expediting investigations of foodborne outbreaks. Ultimately, global efforts contribute significantly to reducing the incidence of foodborne infections.

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