In the present study, the first genetic linkage map of the loach Misgurnus anguillicaudatus was constructed with 164 microsatellite markers and a color locus, and it included 155 newly developed markers. A total of 159 microsatellite markers and a color locus were mapped in 27 linkage groups (LGs). The female map covered 784.5 cM with 153 microsatellite markers and a color locus, whereas the male map covered 662.2 cM with 119 microsatellite markers. The centromeric position in each LG was estimated by marker-centromere mapping based on half-tetrad analysis. In 4 LGs (LG2, LG3, LG4, and LG5), the centromere was estimated at the intermediate region. In LG1, LG11, and LG12, the centromere was estimated to shift from the sub-intermediate region to the end (telomeric). The number of these LGs (7) was identical to the collective number of bi-arm metacentric (5) and sub-metacentric chromosome (2) of the haploid chromosome set (n = 5) of the loach. In the other LGs, the position of the centromere was estimated at the end or outside. These results indicate satisfactory compliance between the linkage map and the chromosome set. Our map would cover approximately almost the entire loach genome because most markers were successfully mapped.