Oenothera was documented by its pharmaceutically active ingredients that require prolonged time to upgrade. Selection of enhanced plants needs high variability. Subsequently, gamma radiation mutagenesis was applied for induction and flaring the genetic variations to get hopeful traits. To examine gamma radio-sensitivity of Oenothera species and the extent of induced genetic variability on earliness and morph-agronomic traits in M1, M2 and M3 generations, field experiments were performed through the sequential seasons 2017/2018, 2018/2019 and 2019/2020. Seeds of both species viz. Oenothera biennis L. and Oenothera albicaulis Pursh were exposed to gamma doses (100, 200 and 300 Gy). The results displayed that both species responded to gamma radiation causing genetic variability reflected on markedly elevation of yield of some selected mutants and earliness in others. Morphological and genetic behaviors altered in both species at all mutagen doses among generations. A steady decline in the survival M1 plants percent after 25 days of sowing was observed with doses upsurge for both species, O. biennis recorded lower response to mutagen doses than O. albicaulis. In M2 generation, coefficient of variation was higher at all doses than the control for both species. Selected mutants significantly enhanced in most studied traits compared to the parallel controls predominantly at 100 and 200 Gy that were more effective on upgrading morpho-agronomic characteristics in M2 generation for both species. Some mutants exhibited formerly earliness than control in M2 generation. Moreover, the superior selected mutants were checked in M3 as 12 mutant lines could be utilized as genetic resources for getting novel varieties with high seed yield, oil and protein percentages. Therefore, exploit gamma mutagens at low and moderate doses; 100 and 200 Gy as effective Oenothera breeding strategy to enrich the seed yield and constituents. Besides, extra selection and assess mutant lines under diverse environments at large-scale to attain the uppermost Oenothera varieties.