Bovine leukemia virus (BLV) was detected and genotyped in a population of 201 dairy cattle from central Mexico. Using a commercial indirect enzyme-linked immunosorbent assay (iELISA) kit, 118 polymerase chain reaction (PCR)-positive and BLV antibody-positive samples were identified; the concordance between tests was substantial. A phylogenetic study of 27 partial sequences of the env gene gp30 was performed. Four mutations were detected involving the PXXP motif in the cytoplasmic domain of the transmembrane protein. This study provided evidence of the efficacy of PCR for the detection of BLV and demonstrated the presence of genotype 1 BLV in Mexico.