A rapid high-performance liquid chromatographic (HPLC) method with simple extraction procedure for the determination of tocopherols in seafood was developed. A continuous isocratic elution system was used for the analysis with a mixture of acetonitrile and methanol. Tocopherols were identified by comparison of retention times and peak area values with standard of alpha-, beta-, gamma- and delta-tocopherols. The HPLC technique used showed good performance parameters (linearity r(2) = 0.9999, and relative standard deviation (RSD) <= 12.38%). The total recoveries for each tocopherol were 99.7, 99.9, 99.8 and 99.7% for alpha, beta, gamma- and delta, respectively. This method was then applied to freshwater and seawater fish species to detect their tocopherol contents. Significant differences (P < 0.05) were observed in the content of tocopherol among fish species. The highest content was obtained from shrimp (81.3 mg/kg), while the lowest was found for whiting (9.8 mg/kg).