Larval forms of the genus Hysterothylacium have been previously reported in teleost fish from the North African coasts of central Mediterranean Sea by morphological analysis. In the present study, samples identified morphologically as Hysterothylacium aduncum (n = 62), from Merluccius merluccius, Trachurus mediterraneus and Pagellus erythrinus from different geographical locations of the Tunisian coasts, were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the sequences obtained with those available in public gene databases confirmed that all the samples from the Tunisian coasts belong to a single species, namely H. aduncum. All specimens from the Tunisian coasts showed one indel in position 787 in ITS-2 sequences not reported by any of the previously published sequences from the Atlantic and Pacific Oceans, Adriatic Sea (Mediterranean Sea) and the East Greenland Sea, suggesting the existence of a population-specific pattern exhibiting a low differentiation of this parasite in this area. This is the first molecular characterization of H. aduncum from the Tunisian coasts using ITS rDNA sequences which allows the definition of genetic markers for their unequivocal identification, and provides further biological data on these nematodes in marine fish off the Tunisian coasts, improving the picture of the occurrence of these taxa in the North African coasts of central Mediterranean Sea.