Vigna is a genus of approximately 100 wild species found in tropical and sub-tropical region. The three varieties of green gram (Vigna radiata L.) (VBN3, VBN6 & CO8) was widely cultivated in Tamil Nadu. Soil salinity is a significant issue in food production because excessive salt concentrations in soil inhibit plant development, delaying maturity and reducing crop yield or causing wilting. In the present study, we investigate the effective screening of saline tolerance in green gram, carried out the mechanism of physiological, biochemical and genetic variation using molecular markers of salinity stress of green gram under different concentration of NaCl (control, 50, 100, 150 and 200 mM). The result shows that, the salinity stress significantly reduced the physiological, biochemical and plant growth characteristics. Besides, the gene-targeted like Start Codon Targeted (SCoT) and CAAT box derived polymorphism (CBDP) markers were used to examine the genetic variability under saline stress of green gram. Herein, the SCoT and CBDP primers produced 54 and 90 polymorphic bands with an average of 5.7 and 7.8 bands per primer, respectively. The Analysis of Molecular Variance (AMOVA) and Principal Coordinate Analysis (PCoA) revealed genetic diversity within species was greater than between them, indicating that all of the saline-treated samples from the three varieties displayed genetic variation. The present study concludes that the increased concentration of NaCl reduced the plant growth and significantly affects the physiological, biochemical and genetic variation.