The widespread use of antibiotics for treatment of bacterial infections and growth promotion in the poultry industry has effectively increased antibiotic resistance around the world. Antibiotics resistance can be caused by different mechanisms and can be determined through phenotypic and molecular methods. The aim of the present study was to determine the occurrence of antibiotic resistance in Salmonella serovars isolated from layer chickens and rats in poultry houses. Phenotypic testing of antimicrobial resistance was performed using the Kirby-Bauer disc diffusion method. Furthermore, molecular evaluations and PCR assay were conducted for detecting resistance genes and class 1 integrons. A total of 144 Salmonella isolates (68 from rats and 46 from chickens) serovars were assessed. Evaluation of phenotypic resistance patterns demonstrated that Salmonella isolates have the highest antibiotic resistance for rifampicin (100%) followed by tetracycline (68%), ciprofloxacin (48%), sulphonamides (42%), chloramphenicol (39%), nalidixic acid (33%), ampicillin (28%), cephalothin (18%), streptomycin (18%), amoxicillin-clavulanic acid (6%), enrofloxacin (5%), and gentamicin (4%). Some Salmonella serovars revealed multi-drug resistance for up to four different antibiotics. According to PCR results, all the tested resistant gene markers (tet, cat, blaTEM, sul, qnrA, and aadA) were detected from the Salmonella isolates. The study further confirmed that 68% of Salmonella isolates were harboring class 1 integrons and the majority of the isolates (n=52) which were harboring these genes were recovered from the rats. The results of the present study provided that the Salmonella spp. isolated from chickens and rats in poultry houses, exhibited significant antibiotic resistance. Moreover, the current research ultimately highlights the importance of rats as carriers of antibiotic-resistant bacteria and the risk of transmission to chickens and humans. DECLARATIONSAuthor's contributions Ramatla T. performed the experiments and wrote the first draft. Moeti OT and Thekisoe OMM provided the analysis tools and data analysis and reviewed the manuscript. Michelo S conceived and designed the experiments, provided reagents, materials and approved the final paper. Consent to publishAll the authors agreed to publish the manuscript and declared that this work has not been previously published elsewhere.