Simple SummaryBlack flies, also known as buffalo gnats, are major pests to humans and animals. Females of some black fly species serve as vectors for transmitting several pathogens (i.e., filarial nematodes, blood protozoa, viruses, and bacteria) to humans and animals via their bites. In Thailand, some human-biting species are considered as natural vectors of zoonotic onchocerciasis. This study was the first to contribute baseline data on the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical forests of the highest mountain in northern Thailand, Doi Inthanon National Park, by using malaise traps. Adult black flies were captured monthly at low to high elevation sites, using malaise traps across three seasons during a one-year period. A total of 44 species were identified among 9406 specimens. It was found that species richness was greatest at the mid elevation. Black fly populations peaked in the rainy season at all elevation sites. The findings of this study showed that varied elevations and seasons are important factors that influence the distribution and abundance of black flies in this region.Black flies form a group of small blood-sucking insects of medical and veterinary importance. This study aimed to investigate the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical rain forests, by using malaise traps in Doi Inthanon National Park, northern Thailand. Malaise traps were placed along six elevational gradients (400 m to 2500 m, above sea level) at Doi Inthanon National Park, Chiang Mai province, from December 2013 to November 2014. A total of 9406 adult female black flies belonging to five subgenera—Daviesellum (2%), Gomphostilbia (23%), Montisimulium (11%), Nevermannia (16%) and Simulium (48%)—were collected. Among 44 taxa found, S. tenebrosum complex had the highest relative abundance (11.1%), followed by the S. asakoae species-group (9.6%), the S. striatum species-group (7.7%), S. inthanonense (6.6%), S. doipuiense complex (6.4%), S. chomthongense complex (5.3%), S. chumpornense (5.1%) and S. nigrogilvum (4.1%). Two human-biting species—S. nigrogilvum and species in the S. asakoae species-group—were found in all of the collection sites with 100% species occurrence. Species richness was highest at mid elevation (1400 m), which is represented by 19 black fly species. The peak and lowest seasonal abundance was observed in the rainy and hot season, respectively. Seasonal species richness was highest in the cold season, except for that from elevation sites at 700 m, 1700 m and 2500 m. This study revealed that the malaise trap is effective in providing important data for further monitoring of the effects of environmental changes and conservation planning on the biodiversity of black flies in Doi Inthanon National Park.