Anopheles gambiae sensu lato complex (An. gambiae s.l.) describes a group of nine morphologically indistinguishable members that vary in their distribution, ability to transmit malaria, and susceptibility to pyrethroids. Here, we recorded the spatial patterns of PCR-identified An. gambiae s.l. complex species collected from four sites in Cross River State, Nigeria that represented three different ecological zones. Trapping was conducted between October 2015 and June 2016. Anopheles gambiae s.l. complex species identification was performed using species-specific primers followed by An. gambiae and An. coluzzii differentiation using the restriction fragment length polymorphism (RFLP) method. Bivariate and multivariate logistic regression models were used to identify ecological and seasonal variables closely associated with An. coluzzii and An. gambiae distribution. Out of 1,388 An. gambiae s.l. successfully amplified, 1,074 (77.4%) were An. coluzzii, 278 (20%) were An. gambiae, and 25 (1.8%) were hybrids (An. coluzzii/An. gambiae). A very small number of An. arabiensis (0.8%, n = 11) were also collected. Statistical analysis indicated that An. coluzzii is predominant in Guinea-savannah and tropical rainforest, and is highly associated with rainy seasons, while, An. gambiae is prevalent in mangrove swamp forest during dry seasons. Only 13 An. gambiae s.l. females were infected with Plasmodium falciparum (P. falciparum). The sporozoite infection rate was higher in mangrove swamp forest (53.8%, n = 7) than in rain forest (38.5%, n = 5) followed by Guinea-savannah (7.7%, n = 1) ecological zones. These results provide important insights for strategic planning of malaria control programs in Nigeria.