We conducted a comparative chromosomal analysis of 10 nominal species and 3 unidentified species in the Simulium striatum group from six countries. A total of 66 chromosomal rearrangements were found, of which 78.8% were inversions. The group is defined by 11 fixed inversions, of which 6 are unique, supporting the monophyletic status previously indicated by morphological and molecular characters. Only 1 of the 13 taxa had a unique fixed chromosomal rearrangement. Although the group demonstrates significant macrogenomic reorganization, subsequent speciation occurred largely without chromosomal rearrangement. The results conflict with the pattern seen in all other species groups of Simuliidae, in which one or more diagnostic rearrangements are typically expressed among species in the same group. The chromosomes provide limited evidence that four entities are valid species but no evidence for the nine others. The weight of evidence from combined chromosomal, molecular, and morphological data, in addition to the practical considerations made for insular species, supports the species status of seven of these nominal species; the remaining taxa require further study. The S. striatum group, accordingly, is either over-divided into nominal species or deficient in chromosomal discriminators. If most or all nominal species and unnamed species are valid, all but one are homosequential, an unprecedented condition in Simuliidae. This group illustrates the need for the integration of multiple character sets for discovering and delimiting species.