Samples obtained from 11 teleost fish species collected in waters off of the Mediterranean coasts of Sicily, Italy, were examined for the presence of Hysterothylacium spp. larvae. In total, 3,017 fish samples were examined, and the larvae recovered were used in subsequent phylogenetic studies. Fifty-eight raphidascaridid parasitic nematodes were found in the examined fish, with prevalence values ranging from 0.2% in anchovies to 60% in forkbeard samples. Twenty-seven parasites were identified as Hysterothylacium fabri and Hysterothylacium aduncum by sequencing the following regions: the nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial cytochrome c oxidase subunit II (COXII). Two other larvae collected from Lophius piscatorius had very low identity scores, with ITS and COXII sequences reported in GenBank and high genetic distances (AY603539). Pairwise comparisons between the ITS region of the H. fabri isolated from fishes from Sicilian coastal waters and those isolated from the Mediterranean Sea, Turkey (KC852206), revealed genetic differences ranging from 0.015 to 0.018. Our H. aduncum samples had very low genetic differences to H. aduncum from the Adriatic Sea (KP979763, 0.00–0.003). The concatenated phylogenetic examination of the ITS-COXII sequences by using maximum likelihood analyses indicated 3 distinct clades supported by high bootstrap values. Further molecular identification and detailed morphological analyses are needed to clarify these results and confirm the diversity and relationships within Hysterothylacium.