Environmental pollution from heavy metals (HM) poses serious threats to aquatic lives. Fish are particularly vulnerable to sub-lethal doses of HM and may lead to genetic mutations and long-term ecological impacts. The study assessed the cytogenetic effects of sub-lethal doses of HM—cadmium (Cd), copper (Cu), iron (Fe), and lead (Pb)—on Oreochromis niloticus from Uta Ewa Creek, receiving aluminium effluent in southeastern Nigeria. O. niloticus samples were exposed to three different sub-lethal concentrations (25, 50, and 75%) of Cd, Cu, Fe, and Pb for 14 days, while a group (control) was unexposed. Random amplified polymorphic DNA (RAPD-PCR) was performed using four polymorphic markers (with 60–70% polymorphism) to evaluate DNA from all fish groups and to identify genetic mutations associated with HM exposure. Genetic differentiation (Gst) in RAPD markers was also examined. RAPD profiles revealed increasing DNA damage in O. niloticus as HM concentrations rose. Cd showed the highest genotoxicity, followed by Cu, Fe, and Pb, compared to control. The control group had 100% GST yield, Pb samples had 30.72% while Cd had 4.33%. These indicated that O. niloticus bioaccumulate these HM with all tested doses causing significant alterations to blood DNA nucleotide sequences. Thus, Uta Ewa Creek contains HM at levels that are genotoxic to local fish populations. Therefore, the efficacy of RAPD-PCR as a tool for detecting genotoxic effects of environmental pollutants in aquatic species was affirmed and there is need for monitoring and regulating metal pollution in aquatic ecosystems to protect biodiversity and ecological health.