Plankton is important for water reservoirs as they are essential for web chains and control populations of bacteria and organic particulates by their filter-feeding behavior. Generally, toxicity tests evaluate mortality or reproduction, however, comet assay has emerged as a useful tool to assess adverse effects on aquatic organisms such as genotoxicity. The present study aimed to value the genotoxic effect of three heavy metals on three zooplanktonic species. Comet assay conditions were adapted in order to obtain minimum DNA migration in negative control and maximum sensitivity in treatments with cadmium, hydrogen peroxide, lead, and mercury. DNA damage was evaluated using two different methods based on the percentage of DNA in the tail. According to our results, mercuryinduced the greatest DNA damage on the three test organisms while cadmium and lead exhibited low genotoxicity. Euchlanis dilatata showed the highest sensitivity to mercury followed by the ciliate and the cladoceran. The comet assay is a liable and consistent technique for DNA damage screening in planktonic organisms. To date, this is the first report for genotoxicity assessment using metals on a rotifer, which represents a reference for evaluating genotoxicity on planktonic species poorly studied.