Consumption of shellfish can yield multiple allergenic reactions in hypersensitive persons. Among shellfish, crab including mud crab can cause mild, moderate, severe and potentially life-threatening allergic reactions. Tropomyosin, a myofibrillar protein at 36 kDa has been recognized as the major allergen in orange mud crab Scylla olivacea by proteomics approach. However, report on the molecular characterization of tropomyosin from S. olivacea and comparative analysis with other crab and crustacean species are unavailable. Hence, this study aimed to clone, identify and compare the molecular structure of tropomyosin in S. olivacea and other crab and crustacean species. This study has first cloned the full-length tropomyosin cDNA from S. olivacea. The sequence was then compared with other custacean tropomyosin in the database. The tropomyosin cDNA of S. olivacea contained an open reading frame (ORF) of 855 bp which encoding a tropomyosin with 285 amino acids that shared high sequence identity with other crab tropomyosin. This includes 99% identity to the giant mud crab (Scylla serrata) and 97% identity to both Japanese blue crab (Portunus trituberculatus) and swimming crab (Charybdis feriatus). While, the similarity with other crustacean species was ranged between 90.14 to 95.74%. As a conclusion, this study demonstrated the sequence of amino acid in tropomyosin of crab and other crustacean species is highly conserved, which might contributing to highly allergenic cross-reactivity between crab and other crustacean species.