| Summary: | Non-typhoidal <i>Salmonella</i> (NTS) is a foodborne pathogen and a prevalent causative agent for disease outbreaks globally. The <i>Salmonella</i> enterica serovar 4,[5],12:i:- (S.4,[5],12:i:-) belongs to the monophasic variant of <i>Salmonella typhimurium</i>, which is of current global concern. In this study, the epidemiology and genomic characterization of S. 4,[5],12:i:- isolates from 17 livestock farms in Hunan Province between 2019 and 2020, as well as their susceptibility to 14 antimicrobial agents, were profiled. Twelve <i>Salmonella</i> serotypes were identified using the White–Kauffmann–Le Minor scheme, and whole-genome sequencing analyses were conducted based on these isolates. Overall, 107 <i>Salmonella</i> strains were isolated, of which 73% (78/107) were multidrug resistant. Resistance to tetracycline (85.05%) was found to be the most prevalent, followed by the <i>oqxAB</i> and <i>aac(6′)-Ib-cr</i> genes. <i>S. typhimurium</i> (monophasic) 4,[5],12:i:- was the most common serotype, followed by <i>S. typhimurium</i> and <i>S. derby.</i> Most antimicrobial-resistant strains were isolated from pigs, indicating that they could be important reservoirs of resistant non-typhoidal <i>Salmonella</i> strains. The presence of similar genetic environments in S. 4,[5],12:i:- indicates both vertical and horizontal transmission of resistance plasmids, which may promote the spread of drug resistance genes. Appropriate measures should be taken to curb the prevalence of S. 4,[5],12:i:-.
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