SummaryBackgroundTilapia is among the most common fresh water fish species raised by fish farms and can cause allergic reactions upon ingestion.ObjectiveTo investigate important allergens in Tilapia (Oreochromis mossambicus).MethodsAllergens were detected using immunoblotting. An important allergen was purified to homogeneity by reversed‐phase High Pressure Liquid Chromatography and characterized by enzyme linked immunosorbent assay (ELISA), competitive ELISA, Mass spectrometry (MS), circular dichroism measurements and differential scanning calorimetry.ResultsBy immunoblotting using sera from 10 patients with confirmed tilapia allergy, we identified a number of allergens with apparent molecular weights 114 to 17 kD. All patients produced IgE against a 32 kD allergen, Ore m 4, which was identified by MS as tropomyosin (TM). IgE binding of the pure protein was confirmed by immunoblotting, ELISA and ELISA inhibition. cDNA from tilapia tropomyosin (TM) was sequenced and compared with TMs from other species. The tilapia TM showed 53.5% homology to TM from shrimp. Homology was much higher to human TM isoform 5 (87.7%).Conclusion and Clinical RelevanceTMs are the major allergens in allergy to crustaceans. Auto‐antibodies against human TM isoform 5 have been implicated as a causative agent in inflammatory bowel disease (IBD). Intriguingly, six of the 10 tilapia allergic patients had also been diagnosed with IBD, corroborating a connection between allergy and IBD. To our knowledge, this is the first report of tropomyosin from vertebrates as an allergen.