Mutants with unique characters have played a key role in discovery of gene, mapping, functional genomics and breeding in many vegetable crops, but information on bitter gourd is lacking. Induction of mutation by gamma rays (Co⁶⁰ source) at five different doses (50 Gy, 100 Gy, 150 Gy, 200 Gy and 250 Gy) was studied in four widely divergent bitter gourd genotypes BG-1346501, Meghna-2, Special Boulder and Selection-1 in M₁ generation. Reduction in seed germination percentage, vine length and pollen fertility occurred in M₁ generation with the increasing doses of mutagens. LD₅₀ dose for BG-1346501, Meghna-2, Special Boulder and Selection-1 corresponded to 290.76 Gy, 206.12 Gy, 212.81 Gy and 213.49 Gy Ƴ radiation, respectively suggested low to medium doses (200–250 Gy) of gamma rays would be helpful in producing useful and exploitable mutants for further breeding. No remarkable effect of Ƴ radiation on fruit physicochemical characters in M₁ generation were observed. M₂ generation, raised from two widely divergent genotypes, BG-1346501 and Meghna-2, were screened critically and observed no significant reduction in seed germination and pollen viability, however little damage occurred particularly in vine length. There is possibility of isolating segregates in M₂ generation with enhanced nutrient contents at lowradiation dose. Highest mutation frequency resulted by treating Meghna-2 at 200 Gy and BG-1346501 at 100 Gy. Both genotype and mutagenic doses influenced mutagenic effectiveness. Spectrum of mutation was very low; number of putative mutants isolated from M₂ generation was five in Meghna-2 and three in BG-1346501. Among six putative macro mutants isolated from M₃ generation, we could identify two putative mutants, namely Meghna-2 with gynoecious sex form and BG-1346501 with high charantin, appreciable ß-carotene and high ascorbic acid contents having ample promise for further utilization in bitter gourd breeding after critical testing in subsequent generations for estimation of genetic gain and trait heritability to confirm the mutant stability.