During frying, fat degrade and many reactions occur resulting in numerous altered fatty acid products. This would lead to the formation of Tran's fatty acids and changes in nutritional qualities. Hence, this study was aimed to determine the changes during frying on fatty acid composition of Nile tilapia (Oreochromis Niloticus) fish muscle from local fish market of Hawassa, Ethiopia. Fish fryers reported that they usually use palm oil for frying. They also indicated that the oil is kept for days and reused for frying at different cycle. In fried fish, 35 types of fatty acids were identified and 27 in raw fish muscle. Palmitic, stearic, heptadecanoic, and tetradecenoic acids were the abundant saturated fatty acids in both fried and raw fish muscle. Oleic, Docosahexaenoic, Eicosapentaenoic, and Linoleic acid were the major unsaturated fatty acids. The percentages of total saturated fatty acids (SFA) of raw fish muscle (47.4%) were found to be lower compared with fried fish muscle (51.8%). The n‐6/n‐3 ratio (7.83), index of atherogenicity (0.85), and thrombogenicity (1.71) in the fried fish muscle were in the undesirable values. The hypocholesterolemic/hypercholesterolemic ratio was relatively similar in the fried (1.09) and raw (1.02) fish muscles. The total unsaturated fatty acids (UFA) level of raw fish muscle (52.2%) was higher than the fried fish muscle (47.2%). Higher percentage of Tran's fatty acid was identified in the fried fish muscle compared with raw. Frying considerably altered fatty acid composition of fish muscle. It increased saturated fatty acid concentration and decreased unsaturated. Hence, frying noticeably decreases the nutritional quality of fish muscle. Therefore, it is suggested that further analysis on fatty acid composition of oil usually used for frying and the nutritional quality index across frying cycles.