Fenugreek (Trigonella foenum-graecum L.) warrants focused attention by researchers to enhance its value and products. The present article investigates how different gamma irradiation doses (25, 50, 75, 100, and 200 Gy) from cobalt 60 influence biochemical parameters of fenugreek and DNA fingerprinting following exposure to irradiation. Irradiated and unirradiated seeds were grown in the field for two successive seasons, and the impact of the applied treatments on photosynthetic pigments (chlorophyll a, b, and carotenoids) and biochemical parameters, particularly total protein, total amino acids, DPPH radical scavenging activity, total flavonoids, and total phenolics, was examined. Results revealed that the 75 Gy dose exhibited a stimulatory influence on chlorophyll content, carotenoids, and biochemicals, whereas the 200 Gy dose exhibited an inhibitory influence on the same traits. Eleven SCoT primers produced 131 markers, including 67 monomorphic, 48 polymorphic, and 16 unique markers with a mean of 46.91% polymorphism. Polymorphic information content (PIC) varied between 0.30 for the SCoT-1 primer and 0.06 for the SCoT-10 primer. In comparison with the control plants, the results exhibited substantial changes in SCoT profiling in the M2 generation due to γ-irradiation treatments and reveal the significant impacts of lowered gamma irradiation dosages, particularly enhancing chlorophyll content and biochemical parameters in fenugreek plants.