Studies were conducted to characterize larval habitats of anopheline mosquitoes and to analyze spatial heterogeneity of mosquito species in the Suba District of western Kenya. A total of 128 aquatic habitats containing mosquito larvae were sampled, and 2,209 anopheline and 10,538 culicine larvae were collected. The habitats were characterized based on size, pH, distance to the nearest house and to the shore of Lake Victoria, coverage of canopy, surface debris, algae and emergent plants, turbidity, substrate, and habitat types. Microscopic identification of third- and fourth-instar anopheline larvae did not yield any Anopheles funestus or other anophelines. A total of 829 An. gambiae s.l. larvae from all habitats were analyzed further by rDNA-polymerase chain reaction to identify individual species within the An. gambiae species complex. Overall, An. arabiensis was the predominant species (63.4%), and An. gambiae was less common (31.4%). The species composition of An. gambiae s.l. varied significantly among the sampling sites throughout Suba District. The larval habitats in the southern area of the district had a higher proportion of An. gambiae than in the northern area. Multiple logistic analysis did not detect any significant association between the occurrence of anopheline larvae and habitat variables, and principal component analysis did not identify key environmental factors associated with the abundance of An. gambiae. However, significant spatial heterogeneity in the relative abundance of An. gambiae within the Suba district was detected. When the effect of larval habitat locality was considered in the analysis, we found that the distance to the nearest house and substrate type were significantly associated with the relative abundance of An. gambiae. Future studies integrating detailed water chemistry analysis, remote sensing technology, and the ecology of predators may be required to further elucidate the mechanisms underlying the observed spatial variation of anopheline larval distribution.