Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffi, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str. and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C + A. simpler s. str., South Africa and New Zealand for A. simplex C + A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (D-Nei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average D-Nei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average D-Nei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of generic variability with respect to both A. simplex s. str. and A. pegreffi, e.g., expected mean heterozygosity, H-e = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str and A. pegreffii appear to belong to distinct food webs, bentho-demersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.