To determine the geographic origin of the black fly Simulium suzukii on Okinawa Island, Japan, macrogenomic profiles derived from its polytene chromosomes were compared with those of mainland and other insular populations of S. suzukii and of the isomorphic Simulium tani species complex. The Okinawan population is a chromosomally unique cytoform, designated 'D,' which is essentially monomorphic and differs by about 27 fixed rearrangements from the chromosomal standard sequence for the subgenus Simulium and by two fixed differences from its nearest known relative, representing the type of S. suzukii, on the main islands of Japan. Chromosomal band sequences revealed two additional, sympatric cytoforms of S. suzukii, designated 'A' and 'B,' each with species status, in Korea, and a third cytoform, designated 'C,' on Hokkaido, Japan. A new cytoform, 'K,' of S. tani from Malaysia, representing the type of S. tani, is more closely related to cytoforms in Thailand, as are populations from Taiwan previously treated as S. suzukii but more closely aligned with S. tani and newly recognized as cytoform 'L' of the latter nominal species. Rooting of chromosomal band sequences by outgroup comparisons allowed directionality of chromosomal rearrangements to be established, enabling phylogenetic inference of cytoforms. Of 41 macrogenomic rearrangements discovered in the five new cytoforms, four provide evidence for a stepwise origin of the Okinawan population from populations characteristic of the main islands of Japan. The macrogenomic approach applied to black flies on Okinawa Island illustrates its potential utility in defining source areas for other species of flies including those that might pose medical and veterinary risks.