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It has learned that soybean is being affected by a floral disorder known as floral malady where plants fail to develop pod and do notattendfull maturity. For this floral disorder, we present a new methylationsensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics of non-methylation, hyper-methylation, hemi-methylation, and full methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied a technique to analyze alterations in the cytosine methylation a popular Indian soybean (Glycine max L.) genotype, JS-335.The result revealed that in the symptomatic plant, out of 392 MSAP sites, 281 (71.68%), 33 (8.41%),38 (9.69%), and 40(10.20%) HIGHLIGHTS• Cytological study for floral malady in soybean• MSAP profiling in floral malady symptomatic and asymptomatic plant.• Insights of pattern and extent of methylation.• Epigentic gene regulation by MSAP approach.