The goal of this study was to isolate and identify the wild yeast strains in intestine of Othius punctulatus collected in Cheongju-si, Chungcheongbuk-do, Korea and characterize their unrecored yeast strains. We isolated 16 wild yeast strains from 5 intestine samples from Othius punctulatus. The 16 wild yeast strains were included three strains in the genus Aureobasidium, four strains in the genus Cystofilobasidium, one strain in the genus Mrakia, three strains in the genus Naganishia, two strains in the genus Saitozyma, two strains in the genus Sampaiozyma and one strain in the genus Scheffersomyces. Among them Cystofilobasidium capitatum YP53, Cystofilobasidium capitatum YP71, Cystofilobasidium macerans YS620, Cystofilobasidium macerans YS622, Mrakia aquatica YP158 and Scheffersomyces stipitis YI5 were recorded for the first time in Korea. The microbiological characteristics and carbon assimilation of these previously unrecorded yeasts were investigated. Almost all of them were oval-shaped. And, glycerol, L-arabinose, xylitol, and inositol are not utilized.