Snakebite envenoming is a major yet neglected tropical disease in sub-Saharan Africa, where antivenom efficacy is critically limited by intraspecific venom variation shaped by local'eco-logical pressures. Nigeria’s sharply contrasting Sudan Savanna (North) and Lowland Rainforest (South) provide an ideal natural system toinvestigate this variation, yet a comparative analysis of its medically important snakes has been lacking. We conducted an integrated proteomic and func-tional characterization of venoms from the puff adder (Bitis arietans) and black-necked spitting cobra (Naja nigricollis) collected in Kaduna (North) and Ibadan (South).Using high-resolution LC-MS/MS, SDS-PAGE, and biochemical assays (PLA₂, protease, fibrinogenolytic, haemolytic, and coagulation activities), we mapped region-specific venom compositions and corresponding toxic activities. Bitis arietans displayed striking ecoregion-driven divergence: southern populations were enriched in ser-ine proteases (27.54%), whereas northern venoms were dominated by lectins (25.4%) and distinct SVMP isoforms. Naja nigricollis showed a conserved PLA₂/3FTx backbone, yet southern venoms ex-hibited elevated SVMP III and LAAO abundance. These molecular differences manifested function-ally, with southern B. arietans showing significantly higher protease activity and southern N.nigricollis exhibiting enhanced PLA₂ activity. Principal component analysis suggested geography as a primary determinant of venom phenotype in both species. This work provides the first integrated proteomic and functional comparison of venoms from northern and southern Nigerian populations of Bitis arietans and Naja nigricollis. While based on a limited number of individuals, the observed differences reveal consistent regional trends that shouldbe interpreted as preliminary and hypoth-esis-generating rather than definitive population-level characteristics.