Probable arthropod vectors of avian blood protozoa, Leucocytozoon lovati, were collected in the alpine regions of Japan, the habitats of the host birds of Japanese rock ptarmigan (Lagopus mutus japonicus). Seven alpine regions of Japan, Asahidake, Chogatake, Tateyama, Jiigatake, Norikura, Kitadake, and Senjyogatake were investigated for black fly collection during 2004 to 2007. The collected 490 insects were morphologically identified as six species of female black flies, including Prosimulium hirtipes group (n = 59), Prosimulium mutata (n = 13), Prosimulium yezoense (n = 10), Similium japonicum (n = 359), Similium uchidai (n = 39), and Twinnia japonensis (n = 10). Extracted DNAs from individual black fly species were utilized for the amplification of the partial mitochondrial cytochrome b gene sequences of Leucocytozoon lovati previously reported. Four S. japonicum, two S. uchidai, and two P. hirtipes group studied were positive for the nested PCR among 490 black flies collected (1.6%; 8/490). All amplified sequences from the black flies were completely identical to those of L. lovati previously detected from Japanese rock ptarmigan. Our results suggest that at least three species of black flies, S. japonicum, S. uchidai, and P. hirtipes group, studied in this area could be regarded as potential vectors for L. lovati in the rock ptarmigan. This is the first detection case of Leucocytozoon from black flies of Japan.