Abstract: The aims of this work were to determine the differential characterization of the urea soluble protein components of puffer fish species and to establish a preliminary proteomic database using an immobilized pH gradient two-dimensional electrophoresis (2DE) technique. The puffer fish muscle proteins resolved into 171−260 spots in the 2DE gels, with a pI range of 3−10 and molecular mass range of 7.4−205.0 kDa, following Comassie blue staining. Puffer fish muscle proteins fell in the region with pI values of 3.5−7.0, and molecular masses of 7.4−45.0 kDa were well-resolved and were good for species comparison. The more acidic proteins of lower molecular masses showed species specific characteristics. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic 2DE protein patterns.