Mitochondrial DNA(mtDNA) variation was investigated in 10 geographical strains of Drosophila virilis. On the basis of cleavage site differences with eight restriction endonucleases, site variation appeared two restriction endonucleases, HindIII and HinfI, and also three different genotypes were identified. Total number of base pair in D. virilis mtDNA molecules was estimated to be 15.86 Kbp, and nucleotide substitution rates among three mtDNA genotypes were ranging from 0.0006 to 0.0066 with an average of 0.0044. In 10 geographical strains, length variation thought to be within the A + T rich-region in mtDNA genome was not found. Also, Geographical cline was not found in three mtDNA genotypes.