Background and Aim: Salmonella has been identified as one of the most widely distributed zoonotic pathogens in broiler litter. Multidrug-resistant strains have been isolated from salmonellosis outbreaks, compromising the success of their treatment. This study aimed to isolate and identify Salmonella spp. serovars in healthy broiler litter in Tolima (Colombia), determine their resistance to different antimicrobials, and detect genes associated with β-lactam resistance that could be useful to control Salmonella spp. in poultry. Materials and Methods: In total, 45 broiler litter samples were collected. Salmonella spp. was isolated and identified using selective and differential culture media and biochemical tests. Molecular confirmation of the pathogen was performed with the invA gene and serotyping by Kauffman–White scheme. Antimicrobial susceptibility to 15 antibiotics was determined by Kirby–Bauer method. In cefotaxime-resistant strains, blaCTX-M-F, blaCTX-M-1, blaCMY, and blaTEM genes were evaluated by polymerase chain reaction (PCR). Results: In total, 817 presumptive strains were obtained from xylose lysine deoxycholate and Salmonella Shigella agars and subcultured on xylose-lysine-tergitol 4 and MacConkey agars, from which 150 strains were isolated; 29 of these strains were presumptive for Salmonella spp. after performing biochemical tests and 16 were confirmed by PCR as Salmonella Infantis (15) and Gallinarum (1). All strains were found to be multiresistant to antibiotics, showing three different profiles and isolates resistant to cefotaxime, and the blaCTX-M gene was detected. Conclusion: This is the first study to isolate S. Infantis from broiler litter in Colombia. All isolates exhibited resistance to the evaluated antimicrobials, suggesting the misuse of antimicrobials in small- and medium-sized poultry farms. The presence of Salmonella enterica serovar Infantis is a public health problem. Thus, regular monitoring of poultry litter is recommended, as these bacteria can be transmitted to humans through animal products or contaminated environments.