Simulium is a very speciose genus of the black fly family Simuliidae that includes many important pests of humans and animals. Cytotaxonomic and morphological studies have made substantial progress in Simulium systematics. 16S rRNA and ITS-1 DNA sequence studies have assisted this progress. Intensive multi-gene molecular systematic investigations will, however, be required for a comprehensive understanding of the genus' taxonomy and evolution. Our research was conducted to investigate the relationships of Thai Simulium at the subgeneric, species group and species levels. We also examined the possibility of using mitochondrial DNA sequences to facilitate Simulium species identification. Data were collected from three mitochondrial genes (COI, ND4 and 16S rRNA) and two segments of the nuclear 28S ribosomal RNA (the D1 to D2 and the D4 expansion regions). The subgenera Simulium and Gomphostilbia were monophyletic in most analyses. Nevermannia included Montisimulium but was otherwise monophyletic in multigene analyses. In most analyses, Simulium and Nevermannia were more closely related to each other than to Gomphostilbia which was usually basal. Species groups were generally monophyletic. Within Gomphostilbia, however, the batoense species group was always paraphyletic to the other two species groups found in Thailand. Three species groups in Simulium were not monophyletic. The tendency to gill filament number reduction for some species groups in the subgenus Simulium was associated with a derived position in multigene analyses. Most species were monophyletic with two exceptions that probably represent species complexes and will present difficulties for rapid mitochondrial DNA identification. © Springer Science+Business Media B.V. 2010.