Abstract: This study was designed to validate the feasibility of using native isoelectric focusing electrophoresis (IEF) of water-soluble sarcoplasmic proteins in identifying puffer fish species. Each of the six tested species of puffer fish Lagocephalus wheeleri, L. gloveri, L. lunaris, L. inermis, L. sceleratus, and Takifugu oblongus showed species-specific protein band profiles after the focused gel was treated with Coomassie blue or silver stain. The majority of water-soluble puffer fish muscle proteins fell in the region with isoelectric point (pI) values of 5.85–8.65. Characteristic species-specific protein bands were present in all the three regions of pI 3.50–5.20, pI 5.85-6.55, and pI 7.35–8.15. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic native IEF protein band profiles.