N. pubescens, N. rubra and N. tetragona is presented based on morphology, RAPD, PCR-RFLP and sequence data of the ITS region, chloroplast trnK intron, matK and rbcL gene in the current study. Morphological examinations revealed considerable variation among the species investigated. Of significant interest, from the context of evolutionary genetic studies, are two sympatric races of N. nouchali viz. N. nouchali JD 06 and N. nouchali JD 07, resembling each other in all aspects but showed flower colour polymorphism with blue and white coloured flowers respectively. Furthermore, members of subgenus (subg.) Lotos namely N. pubescens and N. rubra showed white coloured pollen in contrast to yellow pollen of the remaining species. Interestingly, the white coloured petals of N. pubescens exhibited a tinge of pink at the apex, resembling the petals colour of N. rubra. Barring N. caerulea and N. nouchali, RAPD analysis of randomly selected individuals from each species showed low genetic variation. The variability detected for N. caerulea may be attributed to the breeding system followed, suggesting it to be an outcrossing species. Surprisingly, no genetic variation was recorded among individuals of N. tetragona, a critically rare and endangered plant of India, necessitating its immediate conservation plan. PCR-RFLP of the ITS region revealed additional fragments, exceeding the expected size of the ITS region when totalled, in N. alba var. rubra, N.× marliacea, N. rubra and N. pubescens. Sequencing of the ITS region indicated that the extra fragments, manifested as additional signals in the sequencing chromatogram in N. pubescens may be due to random mutations occurring in some of the ITS paralogues. However, recent hybridization and introgression may be the reason for the additional signals depicted in the chromatograms of N. alba var. rubra and N. rubra. Interestingly, matK gene of N. tetragona revealed higher number of nonsynonymous substitutions. Molecular evolutionary analysis indicated that three of these sites may be under mild selective pressures. Such adaptive changes at the DNA and protein sequence level of matK gene may have been associated with the colonization of N. tetragona, suggesting that it could have migrated from China. Based on neighbour joining, maximum parsimony, maximum likelihood and Bayesian inference, phylogenetic relationship among the investigated species is presented and discussed.