The study aims to perform, for the first time, the molecular identification of anisakid larvae in commercial fish from the Southeastern Pacific Ocean off the Peru coast, and to provide data on their infection level by fishing ground, fish host, and site of infection. Fish specimens (N = 348) from the northern and the central coast of Peru were examined for parasites. The fish fillets were examined by the UV-press method. Anisakis spp. larvae (N = 305) were identified by mtDNA cox2 sequences analysis and by the ARMS-PCR of the locus nas10 nDNA. Two hundred and eighty-eight Anisakis Type I larvae corresponded to Anisakis pegreffii, whereas 17 Anisakis Type II larvae clustered in a phylogenetic lineage distinct from Anisakis physeteris deposited in GenBank, and corresponding to a phylogenetic lineage indicated as Anisakis sp. 2, previously detected in fish from both Pacific and Atlantic waters. Anisakis pegreffii was found to infect both the flesh and viscera, while Anisakis sp. 2 occurred only in the viscera. The average parasitic burden with A. pegreffii in the examined fish species from the two fishing grounds was significantly higher than that observed with Anisakis sp. 2. The results obtained contribute to improve the knowledge on the distribution and occurrence of Anisakis species in Southeastern Pacific waters and their implications in seafood safety for the local human populations.