The European flat oyster (Ostrea edulis) is one of the most significant commercial shellfish species in the Adriatic Sea. Shellfish farming represents the earliest form of mariculture, and for almost a millennium, our country has relied on traditional methods of cultivating flat oysters. One of the reasons for farming is the very high nutritional value of this shellfish species, as well as the favorable ratio of fatty acids and nutrients crucial for human health. In order to carry out an assessment of the chemical composition and fatty acid composition, shellfish samples from the Malostonski Bay area and the mouth of the Krka River were sampled, frozen at -80 °C, and stored at -18 °C and -60 °C, after which analyses were conducted over a period of 3 months. The aim was to determine the chemical changes resulting from preservation by freezing, in order to use the obtained data to improve the production and placement of flat oysters and ultimately the health safety of flat oysters for the market after different periods and temperatures of storage. The results showed that there were no significant differences in the increase of total volatile basic nitrogen (TVB-N) concerning the different freezing temperatures (-18 °C and - 60 °C), and it appears that the pH value of the oysters slightly increased during storage, with a slightly greater increase occurring after the first month of storage as pH values increased by 0,2 to 0,4. The results of the TBA test for determining the degree of fat oxidation showed that the value increased at both storage temperatures, with the overall increase being smaller during storage at -60 °C. Analysis of fatty acids in all samples using gas chromatography with flameionization detector (GC FID) identified 14 different fatty acids, including 6 saturated, 6 polyunsaturated, and 2 monounsaturated fatty acids. In both the flat oysters from Malostonski Bay and the flat oysters from the mouth of the Krka River, in April as well as in May and July, after storage at temperatures of -18 °C and -60 °C, polyunsaturated fatty acids (PUFA) were the most abundant, followed by saturated fatty acids (SFA) and with the lowest proportion being monounsaturated fatty acids (MUFA). Palmitic acid (C16:0) and stearic acid (C18:0) were the most abundant saturated fatty acids, while the most abundant polyunsaturated fatty acids were docosahexaenoic acid (C22:6n3, DHA) and eicosapentaenoic acid (C20:5n3, EPA), and from monounsaturated, oleic acid (C18:1 cis). The proportions of saturated fatty acids in both types of flat oysters mostly slightly increased during storage or had values close to the initial ones at both storage temperatures. In both types of flat oysters and at both storage temperatures (-18 °C and -60 °C), the proportions of polyunsaturated linoleic acid (C18:2 cis) decreased, while the proportions of linolenic (C18:3n3) and docosadienoic acid (C22:2) increased during storage. The greatest change in the proportions of polyunsaturated fatty acids occurred with the decrease in the proportion of linoleic acid in flat oysters at both temperatures, with the greatest decrease occurring during the first month of storage. The proportions of palmitoleic acid (C16:1) increased for monounsaturated fatty acids, while the proportions of oleic acid decreased in both flat oysters and oysters from the Krka River mouth at both storage temperatures. The water content in flat oysters from the Krka River mouth decreased by less than 2 %, while the decrease in water content in flat oysters was less than 1 %. Increases in protein, fat, and ash content were also minimal, with protein content increasing by less than 2 %, fat content by less than 1 %, and ash content by less than 0,3 % in flat oysters from both locations at both storage temperatures (-18 °C and -60 °C).