The highest elevations of tropical mountains, much like oceanic islands, often support disproportionately high levels of endemism, some of which is concealed as cryptic species. In a search for additional species, we explored the chromosomal diversity of three morphologically defined species of black flies known only from the upper elevations of Doi Inthanon, Thailand's highest mountain. Analysis of the polytene chromosomes facilitated the discovery of the previously unknown larva of Simulium kiewmaepanense Takaoka & Srisuka, permitted linkage of the formal name of S. undecimum Takaoka, Srisuka & Saeung with its previous cytological identity, and revealed cryptic species, designated A and B, within S. tenebrosumTakaoka, Srisuka & Atiporn. A probable new species of microsporidium was discovered in the larval fat body of S. kiewmaepanense, suggesting that at least some symbiotic organisms of endemic hosts are also restricted to Doi Inthanon. A mitochondrial DNA analysis did not distinguish the two cryptic species of S. tenebrosum despite complete reproductive isolation chromosomally inferred from an absence of hybrids, emphasizing the need for integrated approaches to species problems.The chromosomes of all four analyzed species were monomorphic, or nearly so, perhaps reflecting the suppression of polymorphic inversions that otherwise would restrict recombination needed to generate adequate variation in the peripheral environment of the mountain summit.