BACKGROUND: Larval stages of the sibling species of parasitic nematodes Anisakis simplex (sensu stricto) (s.s.) (AS) and Anisakis pegreffii (AP) are responsible for a fish-borne zoonosis, known as anisakiasis, that humans aquire via the ingestion of raw or undercooked infected fish or fish-based products. These two species differ in geographical distribution, genetic background and peculiar traits involved in pathogenicity. However, thus far little is known of key molecules potentially involved in host-parasite interactions. Here, high-throughput RNA-Seq and bioinformatics analyses of sequence data were applied to the characterization of the whole sets of transcripts expressed by infective larvae of AS and AP, as well as of their pharyngeal tissues, in a bid to identify transcripts potentially involved in tissue invasion and host-pathogen interplay. RESULTS: Approximately 34,000,000 single-end reads were generated from cDNA libraries for each species. Transcripts identified in AS and AP encoded 19,403 and 10,424 putative peptides, respectively, and were classified based on homology searches, protein motifs, gene ontology and biological pathway mapping. Differential gene expression analysis yielded 226 and 339 transcripts upregulated in the pharyngeal regions of AS and AP, respectively, compared with their corresponding whole-larvae datasets. These included proteolytic enzymes, molecules encoding anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides. CONCLUSIONS: This work provides the scientific community with a list of key transcripts expressed by AS and AP pharyngeal tissues and corresponding annotation information which represents a ready-to-use resource for future functional studies of biological pathways specifically involved in host-parasite interplay.