The objectives of this study were to explore cytogenetic variation and the role of chromosomal change on local adaptation and genetic differentiation of Chironomus circumdatus Kieffer from Thailand. A total of 1,505 larvae from 24 populations were examined cytologically. Twelve chromosomal inversions were found and most of these (9 of 12) were rare inversions. All populations were in Hardy-Weinberg equilibrium. Significant association (P < 0.001) between the A2 and B5 inversions was detected in one population. Population genetic structure analysis indicated significant genetic differentiation between populations (F (ST) = 0.037, P < 0.001). Geographic distance was the principal factor limiting gene flow between populations. Nei's modified genetic distance (D (A)) between populations ranged from 0.001 to 0.011 with an average of 0.003. An UPGMA population phenogram depicting relationship between populations based on D (A) values revealed three groups of populations, group I, II and III each characterized by different inversions/inversion frequencies. Significant correlation of inversion C3 and water temperature suggested that this inversion might have a role to play on adaptation to high temperature habitat. However, if detection of significant population subdivision and relationship between genetic and geographic distance are taken into account, relationship between C3 and water temperature will also be due to the effect of migration/drift alone without the effect of selection.