Clusterbean (C. tetragonoloba) is taxonomically important, underexploited leguminous vegetable crop reported with vast genetic variability. In the present study, an optimized procedure of flow cytometry was used to estimate the genome size between three clusterbean species. Singlet G/G population were determined via flow cytometry using propidium iodide staining dye for a cultivated genotype RGC-936 (C. tetragonoloba) and two wild relatives (C. serreta and C. senegalensis). For accurate estimation of genomic content, multiple tissues i.e. leaves, hypocotyl, and matured seeds and three different plant species viz. Pisum sativum (as primary), Oryza sativa, and Glycine max (as secondary) were used as external and internal reference standards. The genome size of C. tetragonoloba was estimated to be 580.9 ± 0.02 Mbp (1C), which was approximately 1.3 times larger than that of the rice genome. The genome size of C. serreta and C. senegalensis was estimated at 979.6 ± 0.02 Mbp (1C) and 943.4 ± 0.03 Mbp (1C), respectively. In our study, G. max and seed tissue provided the best estimate of nuclear DNA content in comparison to other reference standards and sample tissues used. Findings of this study will enriching genomic database of the legume family and can serve as the starting point for clusterbean evolutionary and genomics studies.