Abstract Background Bovine immunodeficiency virus (BIV) is a member of the Retroviridae family causing a progressive lifelong infection in cattle and buffaloes. Objective Despite the worldwide distribution of the virus, the studies concerning the prevalence of BIV in buffalo populations have not been conducted in Iran as yet. Methods The BIV proviral DNA was surveyed in 120 whole blood samples of water buffaloes in southwestern Iran. Nested PCR was employed to amplify a 298‐bp fragment of the pol gene. The BIV Pol sequence was detected in 9.1% of the samples. Among PCR‐positive samples, two amplified fragments were confirmed by nucleotide sequencing. Results and conclusions The studied sequences were completely identical to each other and had more than 98%–99% nucleotide homology to R‐29 and HXB3 sequences previously deposited in GenBank. Some point mutations that caused coding substitutions were observed in the studied isolates, compared to other strains. A phylogenetic tree was generated based on the BIV Pol nucleotide sequences reported from other countries. All the BIV strains originated from a unique main cluster and then separated from each other over time. This is the first report on the molecular detection of BIV infections in water buffalo populations in Iran. The wide distribution of BIV in different countries including Iran indicates the importance of the infection as it relates to animal health. Although buffaloes show greater resistance to diseases, they should be considered a health risk to cattle. Furthermore, BIV has negative effects on buffalo milk production and can predispose them to secondary infections. Hence, the findings of this study can advance our understanding of the occurrence of BIV infection in Iran, which can play an important role in the distribution of the disease worldwide.