Prosimulium anatoliense new species from western Turkey is described based on the larva, pupa, male, female, and polytene chromosomes. Unique among all known Prosimulium species is the combination of 16 splayed filaments of the pupal gill and the configuration of the teeth of the larval hypostoma. Prosimulium anatoliense is chromosomally unique among Prosimulium species worldwide, but is most closely related to P. hirtipes (Fries) and P. tomosvaryi (Enderlein).