The diversity of Anopheles mosquitoes was studied for seven months in eight villages in Nyabushozi County, Kiruhura District, Uganda. The aim of the study was to identify sibling species of Anopheles gambiae S.L, and the Anopheles funestus group using ribosomal DNA polymerase chain reaction assays. A total of 2566 female A. gambiae S.L, and 270 A. funestus group mosquitoes were collected in 24 households using pyrethrum spray catches, and from goat pens and cattle housing (kraals), using CDC light traps. More A. gambiae S.L, and A. funestus were caught in goat’s houses than in cattle kraals, probably due to the effect of weekly spraying of cattle. The densities of female A. gambiae S. L and A. funestus in all the eight villages studied were significantly influenced by rainfall. A. gambiae S.S. was the only sibling species within the A. gambiae complex identified by polymerase chain reaction assay. A. funestus was also identified by polymerase chain reaction and was found to be in sympathy with two other sibling species within the group, A. nopheles leesoni and A. nopheles parensis. Overall, A. gambiae S.S. was the most abundant species and probably contributed to most of the malaria transmission in Nyabushozi County, Kiruhura district.