In this study, we examined the genetic structure and population history of the high elevation black fly Simulium feuerborni in Thailand at both cytogenetic and molecular genetic levels. Cytological examination revealed two cytoforms differentiated by fixed chromosome inversions. The distributions of the cytoforms were associated with geographic origins. Cytoform A was found in the lower north and northeast, and cytoform B was found in the upper northern region of Thailand. Molecular data based on the mitochondrial cytochrome oxidase subunit I (COI) barcoding sequence supports the separation of the cytoforms. The average sequence divergence between the two cytoforms was 3.75%, which is higher than the threshold value for the species level based on a COI barcoding sequence. Median joining network clearly differentiated the haplotypes of the cytoforms into different lineages. Population pairwise FST and amova analyses reveal significant genetic differentiation between cytoforms. This indicates that the low land areas separating these populations act as a gene flow barrier. No genetic differentiation was detected within cytoforms. This could be due to a recent sharing of population history. Mismatch distribution analysis revealed population expansion in the northern lineage of the cytoform B approximately 220 000 years ago. More recent expansion (32 000 years ago) was found in the lower north and northeast (cytoform A) lineage. The demographic history of S. feuerborni mirrored previous findings in black flies and other insect species in Thailand. This indicates the important role of Pleistocene climatic change on genetic structure and diversity of Southeast Asian mainland species.