Larval forms of the genus Anisakis were reported infecting several fish species from the North African coasts of central Mediterranean Sea. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to investigate the occurrence of larval forms of different Anisakis species in teleost fishes and squid from North African coasts of the Mediterranean Sea and to establish the geographical and host range of these parasites in this area. A total of 282 Anisakis larvae were identified by PCR-RFLP from 13 teleost fish species and one cephalopod species captured at different sites off the Algerian, Tunisian and Libyan coasts. The type I larvae were found with a frequency of 93.62% and were identified as belonging to the following species: Anisakis simplex s.str., Anisakis pegreffii, A. simplex s.str/A. pegreffii hybrids and Anisakis typica. The type II larvae were found to belong to Anisakis physeteris, with the frequency of 6.38%. The record of A. simplex s.str/A. pegreffii hybrids, previously recorded from the Spanish and Portuguese Atlantic coasts and the Alboran Sea, extends their geographic distribution to the Tunisian coasts. The occurrence of A. simplex s.str. and hybrids away from their known area of distribution may predict the successful use of Anisakis larvae for tagging Scomber scombrus fish stocks for fisheries management purposes. Moreover, the results reported provide valuable information regarding the diversity of Anisakis species in the study area, indicating that several Anisakis sibling and morphospecies coexist in the North African coasts of the Mediterranean Sea.