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Four populations of Culex tritaeniorhynchus (Giles) (Diptera: Culicidae), collected from Bellary, Cuddalore, Pune, and the Microbial Containment Complex laboratory culture in India were analyzed for morphological and allozyme variation. Multivariate analysis based on eight morphological characteristics and three morphometric indices was used to investigate the morphological variations among the four populations. Principal component analysis of the data suggested that siphon, saddle, and anal gills related variables were most important. Discriminant factor analysis of morphological data revealed that the four populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, and pectin teeth related variables. Allozyme electrophoresis of the four populations revealed that the mean heterozygosity per locus value had high variation, ranging from 0.0879 to 1.794. Fst values between 0 and 0.519 suggested genetic differentiation within these populations. Fis values ranged from 0 to 1 with most of the values closer to 1. The allelic frequencies and Nei's genetic identity values showed that genetic differences between populations were small, but significant. Some of the morphological and allozyme variations in the Cx. tritaeniorhynchus populations could be partly attributed to the environmental conditions. The findings suggested that transition of morphological characters and allozyme variations in Cx. tritaeniorhynchus populations seem to be consequences of influence and selection by the environmental conditions. These results indicated that populations of Cx. tritaeniorhynchus in non-endemic areas of Japanese encephalitis (JE) virus infection have higher adaptability as compared to endemic areas of JE infection.