Water chestnut (2n = 48) is an aquatic plant of Trapaceae family, the fruits of which are nutritious and have medicinal properties. By selecting from Green Spineless and Red Spineless Biotypes for high TSS and yield from natural population, we developed Improved Red Spineless (IRS) and Improved Green Spineless (IGS) genotypes and registered at N.B.P.G.R as IC 642169 and IC 642170, respectively. These two improved varieties were analyzed with the well-known local varieties using 10 RAPDs and 10 ISSR primers. A total of 78.0 polymorphic fragments with an average PIC of 0.32 and 0.16 and an average MI of 1.48 and 1.09 were produced by the RAPD and ISSR markers, respectively. The dendrogram analysis based on individual and combined RAPD and ISSR markers has demonstrated that the new improved varieties are molecularly distinct from the locally popular varieties. Among four genotypes, the unique gene sequences in 550 bp and 800 bp DNA ladders identified by AP-4 primer and 600 bp DNA ladders identified by AP-32 primer are only found in IRS genotype as dimorphic bands and single band, respectively. Similarly, in IGS water chestnut, DNA fragments AP30-1250, and ISSR23-1500 & ISSR23-1350 are only found as single bands and dimorphic bands, respectively.