ABSTRACT. Sardella C.J. & Luque J.L. [Morphological and molecular diagnostic of Anisakis typica and Anisakis brevispiculata of southeastern coast of Brazil.] Diagnóstico morfológico e molecular de larvas de Anisakis typica e Anisakis brevispiculata em peixes do litoral do Rio de Janeiro. Revista Brasileira de Medicina Veterinária, 38(Supl. 3):87-98, 2016. Programa de Pós-Graduação em Ciências Veterinárias, Anexo 1, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, 23890-000, Seropédica, Rio de Janeiro, Brasil. E-mail: email@example.com The nematodes of the family Anisakidae Skrjabin and Karokhin, 1945 are parasites of aquatic organisms, e.g. fishes, marine mammals and piscivorous birds, being distributed throughout the world. Their larvae are of major importance in the industry of fisheries, since they are known as etiological agents of the human anisakiasis, which occurs through the ingestion of live larvae in raw or poorly cooked seafood. The aim of the present study was to characterize the larvae of Anisakis spp. infecting Brazilian sandperch Pinguipes brasilianus Cuvier, 1829, Frigate tuna Auxis thazard Lacépède, 1800 (fish of commercial importance) and Silvery John dory Zenopsis conchifer Lowe, 1852 in coast of the State of Rio de Janeiro, Brazil, using morphological and molecular approaches. Thirty fish of the species P. brasilianus (Pinguipedidae), 02 of A. thazard (Scombridae) and 10 of Z. conchifer (Zeidae) were analyzed, being collected a total of 04 larvae of Anisakis spp. through the molecular analyzes using mitochondrial and ribosomal DNA sequences and morphological studies, the species Anisakis typica Diesing, 1860 and Anisakis brevispiculata Dollfus, 1966 were confirmed. Zenopsis conchifer representsa new host record for A. typica, and A. brevispiculata was reported for the first time in Brazilian waters.