Abstract: The present investigation deals with the study of, fat, cholesterol and major fatty acid content in river and farm cultivated Labeo rohita (Rohu). Triplicate samples of Rohu were obtained from the river flowing at Doiwala region of Dehradun (U.K) and from the cultivated farm. Fatty acids were determined by conventional biochemical methods, TLC and GC-MS. Fat content of farm cultivated Rohu ranged from 2.5-5 times that of river water samples. Monounsaturated fatty acids predominated in cultivated farm Rohu in comparison to Rohu in river water. Differences in fatty acid profile between river water and farm cultivated Rohu were greatest among polyunsaturated fatty acids; linoleic acid was substantially higher in all cultivated samples than in river water fish. Omega-3 fatty acid levels were also found to be highest in cultivated farm water fish in comparison to river water fish. The study thus indicates that, as per basis of nutritional screening in terms of omega-3 fatty acids, cultivated farm water Rohu are more appropriate for diet.