Tropomyosin is a major allergen responsible for cross-allergenicity in a number of shellfish species. Although extensively characterized in marine crustaceans, the information of tropomyosin is limited to a few freshwater crustacean species. As a result, more cross-reactivity evidence and information of tropomyosin at the molecular level are required for the detection of freshwater crustaceans in the food industry. In this study, we explored tropomyosin allergenicity in four freshwater crustacean species: prawn ( and ) and crayfish ( and ). Immunoblotting, liquid chromatography-tandem mass spectrometry, and immunoprecipitation studies indicated that tropomyosin was recognized by the sera’s IgE of crustacean-allergic volunteers. Cloning and characterization of nucleotide sequences of tropomyosin cDNA from and revealed highly conserved amino acid sequences with other crustaceans. This study emphasized the role of tropomyosin as a universal marker for the detection of both freshwater and marine crustaceans in the food industry.