To elucidate possible causes of skeletal malformations in larval Japanese flounder, we reared larvae fed rotifers enriched with three types of preserved (fresh, refrigerated, and frozen). The incidence of malformations at 50 days post hatch ranged from 14.5 to 38.5 % within the three experimental groups, and elongation of the lower jaw (LJ) was the most frequently observed malformation, ranging from 68 to 89 % of total malformations. We also investigated larval parentages using microsatellite markers. Parentage analysis of the fresh group showed that one sire and a pair generated significant numbers of LJ-elongated individuals. In the refrigerated group, one dam and two sires generated significant numbers of LJ-elongated individuals. In the frozen group, no broodstocks or pairs generated significant numbers of LJ-elongated individuals. Our results suggest that LJ elongation in Japanese flounder likely results from the application of different types of preserved during rotifer feeding stage. However, there is also some level of genetic influence associated with this deformity.