The presence of zoonotic Hysterothylacium larvae in fish from Spanish Atlantic and Mediterranean waters, which can cause economic losses for commercial fisheries, has been reported in several studies; however, little is known about species identity in this region. The aim of this study was to identify at species level the Hysterothylacium morphotypes detected in three commonly consumed fish: horse mackerel (Trachurus trachurus), blue whiting (Micromesistius poutassou) and anchovy (Engraulis encrasicolus). Third- and fourth-stage Hysterothylacium larvae, as well as adults obtained from larval in vitro culture, were morphologically and molecularly identified by ITS1/ITS2 rDNA sequencing. Four Hysterothylacium morphotypes were detected. Genetic analysis showed that morphotypes VIII and IX were different larval stages of Hysterothylacium aduncum, which was supported by cultured adult species identification. Morphotypes III and IV were found to correspond to different developmental stages of another species of Hysterothylacium. As all larval types detected were morphologically indistinguishable from others previously reported yet showed clear genetic differences, they are referred here as new genotypes. This is the first time that ITS-sequence data of various developmental stages of the same species, including adults, have been studied and compared, providing crucial knowledge for future studies on Hysterothylacium identification and biology.