Heavy metal contamination poses a significant risk to aquaculture sustainability and food safety, especially in regions where water bodies serve as major broodstock sources. This study investigated heavy metal contamination in two commonly farmed aquaculture fish species, Oreochromis macrochir and Coptodon rendalli, sampled from six different sites in the Kabompo River, Zambia. Statistical analysis was conducted using two-way ANOVA, with Tukey’s Honest Significant Difference (HSD) test revealing significant differences among the sampling sites and fish species. Nickel (Ni), Zinc (Zn), Copper (Cu), and Manganese (Mn) were detected, while Cadmium (Cd), Cobalt (Co), and Lead (Pb) were below the detection limit. Detected metal concentrations were within permissible limits established by international standards for food safety and potable water, suggesting that heavy metals do not pose significant health risks to fish or consumers. The concentrations of Cu, Zn, and Mn were significantly influenced by both the sampling site and fish species (p < 0.05), with Ni concentrations significantly associated with the sampling site (p < 0.05). No interaction between site and species was observed for any of the detected metals (p > 0.05). Three distinct clusters based on metal concentrations emerged: the Confluence site (Kabompo and Lunga River Confluence), Ntabo, Mubanga, and Mukoka formed one group, while Jivundu and Christella sites formed another. The order of heavy metals across all sites was: Cu > Zn > Mn > Ni. Additionally, physical-chemical parameters of the water, namely dissolved oxygen (DO), pH, temperature, conductivity, salinity, and total dissolved solids (TDS), did not significantly differ across the six sites (p > 0.05) and remained within acceptable limits for tilapia growth. The results of this study are crucial for the development of aquaculture in Zambia, providing baseline data on metal contamination in a key river used for tilapia broodstock and aquaculture water supply. Continued monitoring is recommended to ensure the sustainability and safety of practices in the Kabompo River.