Characterization of the global genetic diversity of the bovine leukemia virus (BLV) is an ongoing international research effort. Up to now BLV sequences have been classified into eleven distinct genotypes. Although BLV genotyping and molecular analysis of field isolates were reported in many countries, there is no report describing BLV genotypes present in cattle from Pakistan. In this study we examined 27 env gene sequences from BLV-infected cattle coming from four farms located in Khyber Pakhtunkwa, Gilgit Baltisan and Punjab provinces. Phylogenetic analyses revealed mthe classification of Pakistani sequences into genotypes G1 and G6. The alignment with the FLK-BLV sequence revealed the presence of 45 mutations, namely, seven in genotype G1 and 33 in genotype G6. Five mutations were found in both, G1 and G6 genotypes. Twelve amino acid substitutions were found in the analyzed sequences, of which only one P264S was specific for sequences from Pakistan. Furthermore, a certain degree of nucleotide heterogeneity was identified by NGS. These results highlight the need for further study on the importance of genetic variability of BLV, especially in the context of its pathogenicity and potential effect on serological detection.