This study was conducted to determine and evaluate the BoLA-DRB3.2 polymorphisms in bovine leukemia virus (BLV) seropositive cows by DNA Sequence Analysis. Isolates of DNA obtained from seropositive (25) and seronegative blood samples (3) collected from cows between 2005 and 2010 were used in this study. DNA isolation was performed from blood samples. MHC analyses were performed using primers belonging to BoLA-DRB3.2 genes. DNA sequence analyses were performed. The number and diversity of haplotypes and nucleotide diversity were calculated. The phylogenetic analysis was determined that cows had 11 haplotypes, healthy individuals were associated together in the first haplotype, while in other haplotypes seropositive cows were associated among themselves and there was 1 seropositive cow between haplotype 6 and 11. Also, the variety of haplotypes (Hd) (0.847), total nucleotide variety (π) (0.033), variation rate (0.00285), and Tajima's D statistical value (0.103) were detected. When F_ST of individuals were evaluated, it was revealed that healthy individuals were genetically distant from positive individuals. In this study, it was observed that BLV seropositive individuals differed genetically compared to healthy animals. In conclusion, BoLA-DRB3.2 gene polymorphism can be considered as a supporting parameter in BLV diagnosis.