Three-hundred eleven tree holes were sampled for Culicoides at 27 localities in Colorado, Kansas, Nebraska, Oklahoma and South Dakota. Of the 311 samples taken, 170 produced specimens, and 2,899 Culicoides were collected including 12 species. The 5 most common species (number collected) were C. guttipennis (1,468), C. arboricola (355), C. paraensis (292), C. elemae (186) and C. footei (159). Other species collected were C. villosipennis, C. nanus, C. hinmani, C. oklahomensis, C. snowi, C. lahillei and C. byersi. New state records include C. byersi and C. arboricola for Colorado; C. arboricola for Wyoming; and C. lahillei, C. elemae, C. nanus and C. paraensis for Oklahoma. Culicoides byersi, whose larval habitat was previously unknown, was reared from a cottonwood tree hole.