The phytophagous ladybird beetle belonging to group B of the Henosepilachna vigintioctomaculata complex includes three closely related species. The northern species, Henosepilachna pustulosa, feeds on both thistle (Asteraceae) and blue cohosh (Berberidaceae). On the other hand, the two southern species, H. niponica and H. yasutomii, are sympatric and depend on thistle and blue cohosh, respectively. Since evolution of different host use directly causes speciation of the latter two specialist species, detection of genome region associating with different host use is indispensable to understand the speciation process of these ladybird beetles. Here, we examined the genomic differentiation responsible for the different host use of the three species. Comparative genome scan using 627 valuable AFLP markers for totally 164 individuals of the beetle reveals that makers associating host use were much more diverged than those of neutral region. The result suggested that evolution of divergent host use facilitates entirely genomic divergence between specialist species via restriction of gene flow. We report the amount of genomic divergence that are associated with different host use and discuss the process of speciation of these species.