In Japanese yellowtail (Seriola quinqueradiata) aquaculture production, Benedenia disease is a serious parasitic disease caused by infection by the monogenean parasite Benedenia seriolae. Quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2. To reveal regulatory mechanisms and responsible gene for Benedenia disease resistance, bacterial artificial chromosome (BAC) clones carrying genomic DNA of QTL region were screened from a yellowtail BAC library. A minimal tiling pathway was constructed by seven BAC clones. These seven BAC clones were sequenced by Illumina sequencing.