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The aim of this study was to determine the influence of different feed composition and cultivation on the composition of fatty acids, their relationships, and the cholesterol content in the tissue of rainbow trout ( Oncorhynchus mykiss). Trout were reared in ponds at two different altitudes and raised on different feeds. The total amount of monounsaturated fatty acids in fillets of both groups showed no statistically significant difference. However, higher levels of n-6 fatty acids were found in Pond I trout fillets than in Pond II fillets, while Pond II fillets contained almost even amounts of n-3 and n-6 fatty acids. Total cholesterol content in Pond I trout fillets was significantly lower ( p < 0.05) than in Pond II fillets. The results indicate that the final composition of the trout fillets was mostly dependent on the feed the live fish had consumed; the altitude of the ponds had less effect. Also, both groups of trout could be used in the human diet as biologically valuable foods. Practical application: Our results show influence of altitude, feed, and fatty acid composition of feed on trout fillets. The average fatty acid level in the trout fillets increased in the order SFA