Anisakid nematodes have been found in a variety of marine fishes throughout the world and they areknown to cause anisakiasis in human hosts. The present study investigated the prevalence of potentiallyzoonotic anisakid larvae in spotted mackerel caught from Taiwanese waters where fish represents animportant food sources. Anisakis third-stage larvae (L3, n = 502) were isolated from 250 spotted mackerelScomber australasicus. Anisakis L3 larvae were divided morphologically into two types, Anisakis type Ilarvae had a longer ventriculus and mucron while type II larvae had a shorter ventriculus and no mucron.Anisakis species were identified by polymerase chain reaction-restriction fragment length polymorphism(PCR–RFLP) of the internal transcribed spacer (ITS) regions of ribosomal DNA and direct sequencing. Asimple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled thedifferentiation of the genus Anisakis. The prevalence, mean intensity and mean abundance of Anisakisnematodes recorded for the total specimens were 72.8%, 2.8 (1–15) and 2.0 (0–15), respectively. Anisakispegreffii was determined to be the dominant species (prevalence = 57.2%) and important agent of humananisakiasis. A recombinant genotype (Anisakis simplex sensu stricto × A. pegreffii) was identified as thesubdominant species (25.3%) followed by Anisakis typica (10%), Anisakis physeteris (4.0%), Anisakis paggiae(3.0%) and Anisakis brevispiculata (0.5%). The topology of the maximum likelihood and neighbor-joiningtrees show two well supported clades: one includes the species of A. pegreffii and the other includes A. pag-giae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed.This study advances our knowledge of the prevalence of different Anisakis spp. in the spotted mackerelfrom Taiwanese waters, which is helpful for monitoring the fish populations throughout a diverse arrayof aquatic ecosystems. More importantly, we provide the concise characterization of multiple Anisakisspp. by PCR–RFLP, which could also be applicable for the rapid diagnosis of human anisakiasis.