Abstract: Berenil (Diminazine aceturate) is an active trypanocidal drug used in farm animals such as cattle and river buffalo. In human, berenil is an effective drug in the treatment of early stage of African trypanosomiasis and cutaneous leishmaniasis. In this work, the cytogenetic effect of berenil was tested in the lymphocyte cultures of river buffalo at three final concentrations; 0.25, 0.50 and 1 mg ml ha-1 . Chromosomal aberrations, sister chromatid exchanges and micronucleus test are the three cytogenetic parameters used in this study. The results demonstrated that the numbers of cells with different types of structural chromosomal aberrations, including breaks, gaps, deletions, fragments and centric fusions were increased significantly in cultures treated with different doses of berenil compared to the control. This increase was dose dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations. The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of berenil were increased significantly compared to the control and these increases were also dose dependent. In conclusion, the three cytogenetic parameters used to evaluate the effect of berenil revealed that this drug has a strong cytogenetic effect on the river buffalo lymphocytes in vitro.