Abstract: The current study was aimed to investigate the genotoxic effect of an organophosphate pesticide malathion in the gills of a freshwater teleost rohu, Labeo rohita using alkaline Single Cell Gel Electrophoresis (SCGE)/comet assay. The 96 h LC50 of malathion was estimated for rohu in a semi-static system and was found to be 5 μg L–1. Specimens of rohu were exposed to LC50 of malathion. Gill tissues were sampled after 24, 48, 72 and 96 h of exposure. DNA damage was evaluated by studying different indices, including tail length (μm), percentage of DNA in tail, tail moment and olive tail moment using TriTek CometScoreTM. A linear relation was observed between exposure time and DNA damage in the gill cells. The current study revealed malathion as a potent inducer of DNA damage and comet assay as a reliable and sensitive assay for investigating and detecting DNA damage in vivo, induced in fish by genotoxic pesticides. In order to conserve the vanishing populations of rohu in natural aquatic bodies across the country, indiscriminate use of genotoxic pesticides such as malathion should be minimized.