Until now, Anisakis typica has been the sole anisakid identified by means of genetic markers from the Atlantic Ocean off Brazil. In this study we developed, and applied to larvae and adults, an A. typica-specific molecular identification protocol based on the complete intergenic transcribed spacer (ITS) region. Anisakids from the dolphin Sotalia guianensis and from the frigate tuna, Auxis thazard, caught off the coast of Brazil, were processed using two DNA extraction methods. Molecular diagnosis identified A. typica in 13 of 15 samples. Complete ITS analysis showed that the remaining two isolates were in fact A. physeteris. Maximum parsimony analysis of complete ITS region (915 bp) confirmed these results. Our study verified the dominance of A. typica infecting hosts from the Brazilian coast and showed, for the first time, the presence of A. physeteris in the intermediate/paratenic host A. thazard in this region.