Next Generation Sequencing technology with an Illumina GAIIx platform was used for the sequencing of a normalized cDNA-AS library genes for the nematode parasite Anisakis simplex (s. l.). A de novo assembly (A1), structural and functional annotation of the transcriptome was undertaken. Subsequelntly, a second reference guide assembly (A2) (Sanger A. simplex, GCA_000951095.1 genome assembly Howe et al., 2016) using the information from the RNA sequencing from the first assembly was also performed. Once the second assembly (A2) was completed, we compared the generated data against the A1 transcriptome. The obtained protein sequences were analysed against the proteomes of invertebrate organisms (UniProtKB - Swiss-Prot). Obtained data were used to identifify through fingerprinting in combination with tandem mass spectrometry new A. simplex (s.l.) allergens. The homologes of the new identify allergens were identified in the public data base of the sibling species Anisakis simplex (s.s.), Anisakis pegreffii and their hybrid.