We examined the genetic structure of populations of an ambrosia beetle, Xylosandrus crassiusculus. Specimens were collected from 22 sites in Japan and their genetic structure was studied using a portion of the mitochondrial cytochrome oxidase I gene. Phylogenetic analysis revealed three distinct lineages (clades A, B and C), each of which was divided into two subclades (A₁, A₂, B₁, B₂, C₁ and C₂). Subclade A₁ had 17 haplotypes from Hokkaido, Honshu and Kyushu populations, subclade A₂ 5 haplotypes mainly from Shikoku populations, subclade B₁ 15 haplotypes from central and western Honshu and Amami populations, subclade B₂ 2 haplotypes only from Naha population, subclade C₁ 4 haplotypes from Amami and Naha populations, and subclade C₂ 5 haplotypes only from Ishigaki population. Analysis of molecular variance detected genetic differentiation among populations. The data imply that the genetic structure of Japanese populations of X. crassiusculus has been affected by the geological history of the Japanese islands, but that the mtDNA phylogeographic pattern is so complex that we could not infer any simple scenario of population history in X. crassiusculus.