Four specimens of the five-gilled white mid-dorsal line hagfish, Eptatretus wandoensis sp. nov. were recently collected from the southwestern Sea of Korea (Wando). This new species has five pairs of gill apertures, 14–18 prebranchial slime pores, 4 branchial slime pores, a dark brown back with a white mid-dorsal line and a white belly. These hagfish are similar to Eptatretus burgeri and Eptatretus minor in having a white mid-dorsal line, but can be readily distinguished by the numbers of gill apertures (5 vs. 6–7), gill pouches (5 vs. 6), and prebranchial slime pores (14–18 vs. > 18), as well as the body color (dark brown back vs. gray or brown pale). In terms of genetic differences, Eptatretus wandoensis could be clearly distinguished from E. burgeri (0.9% in 16S rRNA and 8.5% in cytochrome c oxidase subunit I sequences) and E. minor (4.5% and 13.9%).