The impact of gamma irradiation on fenugreek (Trigonella foenum-graecum L.) for two generations was investigated in this research. The study involved exposing dry seeds of fenugreek to five doses of gamma rays (25, 50, 75, 100, and 200 Gy) using a cobalt 60 gamma radiation source. The irradiated and unirradiated seeds were grown in the field for two consecutive seasons. The results showed significant changes in vegetative growth traits as well as yield parameters in both the M₁ and M₂ generations. Low doses of 25–100 Gy stimulated growth and increased yield in M₁ and M₂ plants, while the highest dose of 200 Gy showed an inhibitory effect on the same parameters. The 75 Gy resulted in the highest increases in all traits. Eleven ISSR primers were used to identify molecular differences in ISSR fingerprinting in response to γ-irradiation treatments in the M₂ plants. The 11 ISSR primers produced 118 markers, revealed as bands on the agarose gel, including 73 monomorphic bands, 26 polymorphic bands, and 19 unique bands with an average polymorphism of 37%. The cluster analysis of ISSR data differentiated the M₂ plants grown from seeds exposed to 75 Gy of gamma radiation, indicating that the highest increase in vegetative and yield traits was associated with polymorphic ISSR markers. This result shows that ISSR profiling is a good way to connect genetic differences that happen in plants of the M₂ generation in response to γ-irradiation treatments.