This study analyzed the expressed sequence tags (ESTs) of the third-stage larvae of Anisakis simplex, in an attempt to gain further insight into its genomic expression patterns. An A. simplex cDNA library was constructed using the Uni-ZAP XR expression vector. A total of 493 clones (insert DNA>400 bp) were sequenced out of 580 clones selected randomly from a cDNA library of the A. simplex third-stage larva. After BLAST search analyses, 154 (31.2%) ESTs were found to have very low similarity, or no match at all to any of the proteins and gene sequences in the published databases. Most matched clones (98 clones, 20.0%) were determined to be highly homologous with the genes or proteins of Caenorhabditis elegans. Ten (2.0%) ESTs matched the genes isolated from humans, and 21 (4.3%) ESTs matched with the previously reported A. simplex genes or proteins. Eighty-nine clones (18.0%) matched a total of 14 genera and 17 species of human parasites. These 339 ESTs identified could be grouped into 13 categories: allergens or antigens (4.1%), growth- and cell division-related proteins (3.2%), heat shock proteins or molecular chaperones (1.8%), membrane proteins (5.6%), metabolism-associated proteins (24.2%), mitochondrial proteins (9.4%), nuclear proteins (2.4%), proteases and protease inhibitors (3.5%), signal transduction proteins (2.4%), structural proteins (7.4%), transcription and translation machinery-associated proteins (20.1%), transporters and receptor proteins (3.8%), and other protein types (12.1%). The genetic information of Anisakis determined in this study might prove to be quite helpful in elucidating the pathogenetic mechanisms of anisakidosis, and might be useful in the development of therapeutic reagents specific to anisakidosis.