Bovine leukaemia virus (BLV) causes enzootic leucosis in cattle and is prevalent worldwide. Although recent studies have shown that BLV strains can be classified into 10 distinct genotypes, no information is available regarding the BLV genotype prevalent in cattle in India. To determine the genetic variability in BLV, in this study, 118 adult dairy cows from three states of India were screened for BLV infection by env gp51-specific ELISA and nested PCR. Of the 33 cows found positive by both PCR and ELISA, 10 selected BLV strains were subjected to molecular characterization. Phylogenetic analyses of partial and full-length env gp51 gene sequences of Indian BLV strains and other geographical diverse BLV strains representing all the 10 genotypes revealed that Indian strains belonged to BLV genotype 6. Although Indian strains showed close genetic proximity with the strains circulating in South America, they were classified into a new subgenotype within genotype 6. Alignment of deduced amino acid sequences in gp51 demonstrated substitutions mainly in conformational epitope G, neutralizing domain 2 and linear epitope D, with a novel mutation (threonine to alanine at residue 252) found in D-epitope of all the Indian BLV strains. Although serological evidence of BLV infection in India has been reported earlier, this study on molecular characterization of BLV strains established the existence of BLV genotype 6 in India. Additionally, the results of this study highlight the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity and further studies are required to determine BLV genetic diversity and extent of BLV infection in cattle in India.