Abstract: The present study was conducted to evaluate the effect of different doses of gamma rays (0, 0.75, 1.5, 2.25, 3, 3.75 and 4.5 kGy) on fatty acids composition of Rainbow trout fillet. Irradiation of fillets was performed by gamma rays from a Co60 source. The results showed that total saturated fatty acid concentrations increased significantly (p<0.001) with increasing irradiation dose, so that the control sample and fish muscles that irradiated with 4.5 kGy had the lowest and highest amounts of total saturated fatty acids (20.454±0.011 and 19.228±0.040%, respectively). The amount of total Polyunsaturated Fatty Acids (PUFAs) in irradiated samples were significantly lower than control sample (p<0.001) and amounts of total monounsaturated (MUFAs) were significantly higher than control samples (p<0.001). The results indicated that the highest content of MUFAs and PUFAs were in samples irradiated with 3.75 kGy (37.783±0.092%) and control samples (37.677±0.104%), respectively. The lowest level of MUFAs and PUFAs were in control samples (36.596±0.024%) and 4.5 kGy (36.459±0.047%), respectively. The results of this study showed that Gamma irradiation influenced the fatty acid composition especially polyunsaturated fatty acids in rainbow trout fillet significantly (p<0.001).