Cytoform C, one of three cytoforms in the Simulium angulistylum Takaoka & Davies complex from a high mountain in northeastern Thailand was morphologically and molecularly investigated. All known life stages (larva, pupa, adult male and female except egg) were morphologically similar to, but distinguishable from S. angulistylum s. str. and S. isanense Takaoka, Srisuka & Saeung in the adults by the relative length of the fore and hind basitarsi and relative length of the tooth to the claw. It is also morphologically distinct from other species of the S. epistum species-group. Here, it is formally described as a new species, S. prayooki. Molecular genetic data based on mitochondrial cytochrome c oxidase subunit I (COI) also supported the morphological similarity between the new species and the two related known species (S. angulistylum s. str. and S. isanense) as phylogenetic analysis retrieved them all from a single clade and with a low level of interspecific genetic divergence (1.74%). This might possibly have resulted from incomplete lineage sorting as they are likely to share a recent common ancestor. Despite limitation of molecular genetic differentiation, the new species was distinctly different from two other cytoforms of S. angulistylum complex based on polytene chromosome banding patterns and ecology of the immature stages. Thus, this study highlights the necessity of using an integrated approach for fully understanding black fly biodiversity.