This study provides means for more accurate identification of Chironomus circumdatus species for environmental and ecological management by Population of C. circumdatus (86 individuals) and water samples were collected from three different sites of Chiang Mai Province, Thailand. Specimens of Einfeldia sp. were used as an initial out group. The genetic relationship of C. circumdatus specimens from these sources was determined by using DNA sequence analysis of partial mtDNA gene’s Cytochrome Oxidase Subunit I (COI). Analysis of genetic distance on the basis of sequence difference for COI mitochondrial gene showed very little genetic difference and the data from phylogenetic analysis revealed a very large genetic difference among all populations of this species for the COI gene sequences. In addition, NJ tree was related to with physicochemical parameters of the water samples.