Bovine mastitis is considered a problem that is impossible to eradicate, due to the indiscriminate use of antibiotics, mainly β-lactams. The aim of this study was to detect antimicrobial resistance genes in bacteria isolated from bovine mastitis in Western Nicaragua. The antimicrobial resistance profile of 30 bacterial strains was evaluated using the phenotypic method for the antibiotics amoxicillin/clavulanic acid (AMC), ceftriaxone (CRO), gentamicin (CN), cephalexin (CL), vancomycin (VA) for gram-positive bacteria, while the antibiotics amoxicillin/clavulanic acid (AMC), trimethoprim sulfamethoxazole (SXT), ceftriaxone (CRO), gentamicin (CN), cephalexin (CL) in gram negative bacteria. In addition, the evaluation of the genotypic method was carried out using the PCR (Polymerase Chain Reaction) technique for the detection of extended-spectrum β-lactamases (ESBL) in gram-negative bacteria and the mecA gene for bacteria of the Staphylococcus genus (MRS). It was possible to identify that the bacterial isolates presented a greater resistance to amoxicillin and oxacillin with 24/30 and 10/14, respectively. On the other hand, 100% of the bacterial strains showed sensitivity to the antibiotics gentamicin (CN) and sulfamethoxazole (SXT). In the present study, 2 antimicrobial resistance genes were detected in the gram-negative isolates related to ESBL, the blaSHV gene coding for the strains: Enterobacter, Serratia and E. coli, while the blaTEM gene was detected in the strains: Enterobacter and Serratia. The blaCTX gene in gram-negatives and the mecA gene in Staphylococcus were not detected.