Anisakid nematodes belonging to the species Anisakis pegreffii are distributed in a wide variety of fishes from the Mediterranean Sea and they are known to cause the human zoonosis anisakiasis. The present study investigated, for the first time, the response of A. pegreffii larvae (identified to species level by allozymes and mtDNA cox2 sequence analysis) to the storage temperature of European anchovies, Engraulis encrasicolus. The larval motility of A. pegreffii was studied in 1300 fish specimens, captured from a highly infested area (FAO 37.2.1, 43°8′N, 14°16′E), maintained under different temperatures (2 °C, 5 °C, 7 °C), and examined at different time intervals (immediately after fishing, 24 h, 48 h and 72 h). Parasitological analysis was carried out with the UV-press method. The results showed that the increase of infection values with A. pegreffii in the fillets of anchovies was statistically positively related to the increase of the temperature (at 5 °C and 7 °C) and time of storage (after 24 h, 48 h, and 72 h). Accordingly, a significant statistical correlation between the increasing of the worm burden in the fillets and a decreasing of A. pegreffii in the viscera was observed. In contrast, those fish constantly maintained at 2 °C showed no statistically significant variation in infection either in the viscera or the fillets, after 24, 48 and 72 h. In the same batches of anchovies, larvae of the non-zoonotic nematode parasite Hysterothylacium aduncum (identified to the species level by ITS rDNA sequences analysis) were found, but they were never observed infecting the musculature of the anchovies. Our results suggest that temperature plays an important role in the post-mortem motility of A. pegreffii larvae in anchovies. In addition, the presence of A. pegreffii in the fillets inspected immediately after their capture indicates that intra-vitam migration may also occur. As a consequence, the importance of the adoption of rules to prevent human anisakiasis, as the deep freezing to −20 °C for 24 h, was underlined. •The response by Anisakis pegreffii to storage temperature in anchovies was investigated.•The increase of A. pegreffii larvae in the fish flesh is positively related to the temperature.•The temperature plays a role in the post mortem motility of A. pegreffii larvae in anchovies.•A. pegreffii in the fillets of anchovies can be also the result of intra-vitam migration.