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This study employs molecular detection techniques, including conventional PCR and Sanger sequencing, to investigate the prevalence, species composition and public health implications of Anisakid nematodes in blue whiting (Micromesistius poutassou) caught off the Portuguese coast. With Portugal’s high fish consumption rates and increasing preference for raw or undercooked seafood, the risk of parasitic infections, particularly anisakidosis, is rising. Fifty blue whiting fish were examined, showing a 100% infection rate with Anisakid larvae. Molecular analysis identified 68.1% of the larvae as Anisakis simplex, 18.1% as Anisakis pegreffii, and 13.8% as Hysterothylacium aduncum, marking the first report of H. aduncum in blue whiting in Portugal. Phylogenetic analysis based on the internal transcribed spacer (ITS) 1, 5.8S ribosomal RNA and ITS-2 confirmed the species classification. Notably, 42.9% of the fish were infected with multiple Anisakid species, increasing the risk of allergenic sensitization. Statistical analysis showed no significant correlation between fish width and parasitic load, and a weak negative correlation was found between fish length and parasitic load. The study contributes to food safety by integrating molecular tools that enable rapid and accurate species identification, offering new insights into the detection of biological contaminants in seafood. These findings are significant considering the rising trend in raw seafood consumption, underscoring the urgent need for enhanced detection strategies and broader parasite monitoring programs to mitigate public health risks. The high prevalence of parasitized fish highlights the necessity for the implementation of safe cooking practices to reduce the risk of anisakidosis. Further research into the allergenic potential of Hysterothylacium spp. and the ecological factors influencing this nematode distribution is recommended.