Aims This study evaluated the antimicrobial resistance of Salmonella enterica strains from layer poultry farms in central Ecuador isolated during 2017. This geographical area is responsible for around 60% of total domestic egg production, yet, as of 2019, no reports had been published on the phenotypic and genotypic antibiotic resistance patterns of Salmonella in the layer poultry farms of this area. Methods and Results Thirty-one isolates from layer poultry farms in central Ecuador obtained during 2017 were evaluated. The resistance profiles exhibited considerable differences in serovar and sample origin, grouping into nine clades by phenotype. S. Infantis strains were of the MDR phenotype in 94 center dot 4% of isolates. S. Typhimurium strains were of a reduced antimicrobial resistance phenotype and 50% showed resistance to one antimicrobial compound. One of the S. enterica nontyped strains had an MDR profile to 11 of the 20 antibiotics evaluated (eight groups). And the two remaining S. enterica nontyped strains showed resistance to two and three antibiotics respectively. The ESBL phenotype, which is resistant to clinically notable antibiotics such as ceftriaxone, ampicillin and cefepime, was observed only in S. Infantis (15/18). These strains harbour the emerging bla(CTX-M-65) gene, and co-harbour tetA and sul1 resistance genes in four strains. Additional beta-lactamase genes, carbapenemase-producing genes (bla(IMP,) bla(VIM), bla(OXA48), bla(KPC), bla(NDM)) and colistin-mobile resistance gene mcr-1 were not detected. Conclusions The findings highlight the potential role of layer poultry farm environments in central Ecuador as reservoirs of MDR Salmonella strains. Significance and Impact of the Study These results suggest the necessity of reinforcing biosecurity practices to reduce the probability of transmission of MDR Salmonella across the food chain.