The polytene chromosomes and larvae of two species of black flies, Simulium gandakiense new species and S. magnacentrum new species, are described from the Himalaya Mountains of Nepal. Twenty-three chromosomal rearrangements were discovered in the two species, of which eight are shared. The species differ by four fixed inversions and by having unique sex chromosomes. Additionally, S. gandakiense has B chromosomes and S. magnacentrum has a chromocenter. Both species have a set of four fixed chromosomal inversions and accessory sclerites in the larvae, all of which are shared with members of the S. vernum species-group in the Oriental Region, suggesting a biogeographic connection between the Himalayas and the Oriental Region for the S. vernum species-group.