Sterol contents (desmosterol, cholesterol, stigmasterol, and sitosterol) of 36 fresh and cooked seafood and four freshwater fish species were investigated by high performance liquid chromatography (HPLC) method. Cholesterol and sitosterol were main sterols in seafood and freshwater fish species. Raw shellfish and molluscs contained cholesterol above 18.92 mg/100 g fish muscle, while the cholesterol content of marine fish ranged from 6.5 to 78.40 mg/100 g fish muscle. The cooking process resulted in significant effects on the sterol contents of seafood and freshwater species (p < 0.05). A remarkable increase in sitosterol (more than 3-4 fold compared to raw fish) was recorded for some fish species cooked in the oven. The highest desmosterol content was observed for fried fish, whereas the frying process resulted in a significant loss in cholesterol and sitosterol contents of marine fish (p < 0.05). The impacts of cooking methods on sterol content of seafood and their form varied depending on fish species and the cooking method used.