<p>The current study explores the impact of gamma radiation on the in vitro morphogenesis of <i>Moringa concanensis</i>. In vitro regenerated shoots were exposed to ¹³³Barium and ⁵⁷Cobalt gamma radiation sources for varying lengths of time (3, 6, 9, and 15&#xa0;min). All the treated shoots survived with 100% regeneration frequency. The number of regenerated shoots was increased to 4.33 ± 1.57/inoculum in cobalt radiation-treated shoots. The field survival rate was increased, and 70% of plantlets from gamma radiation-treated shoots were successfully transferred to polybags. The multiple layers of epidermis, elongated cortical cells, pericycle cells, and increased content of vascular elements were observed in the anatomical assessment of regenerated shoots after treatment. The variations and altered responses of the treated shoots were further evaluated through CBDP (CAAT Box Derived Polymorphism), SCoT (Start Codon Targeted) gene-based, and ISSR (Inter Simple Sequence Repeat) intergenic sequence-based markers. An effective range of polymorphism of 75.00%, 77.77%, and 80.76% was observed from all the employed primers. A total of 0.2 PIC value was obtained from all used 6 primers that represent their informativeness in evaluating diversity among genotypes. The given minimum dose influenced the in vitro growth, anatomical development, and variations in genomic sequences, proving gamma radiation as an effective mutagen for <i>Moringa concanensis</i>. The gamma radiation source ¹³³Ba and ⁵⁷Co would be further used as a physical mutagen for developing efficient varieties of <i>Moringa concanensis</i> for the <i>Moringa</i> breeding program.</p>