1 The tephritid fruit fly Zeugodacus tau is a serious agriculture pest, especially of cucurbits in Thailand, where it is classified as a complex species of nine siblings (Z. tau A, B, C, D, E, F, G, I and J). 2 Based on single-strand conformation polymorphism (SSCP) analysis, 33 distinct mitochondrial cytochrome c oxidase subunit I (mtCOI) haplotypes were found in three sibling species, Z. tau A, C and J, in Thailand. Genetic structure analysis of the population revealed relatively large genetic differences among these sibling species, reflecting a tendency toward speciation. 3 The maximum likelihood tree and median-joining network reconstructed from the mtCOI sequences of 33 haplotypes supported the separation of these three sibling species within the Z. tau complex. 4 The three-level hierarchical analysis of molecular variance based on geographical regions and the Mantel test for isolation-by-distance showed significant genetic structuring of the Z. tau complex in Thailand. 5 However, the genetic differentiation among geographical groups was rather low, which may be reasonably explained by natural and human mediated dispersal, as well as host associated differentiation. 6 The SSCP analysis on the population genetic structure of Z. tau species complex in the present study should benefit pest management.