In this study, we isolated and analysed the sequence of ITS4-5 genetic region of thirty three lotus samples which were collected in Thua Thien Hue province. The attained ITS4-5 genetic region had a length of ranged from 729 to 744 bp, which had high similarity with species Nelumbo nucifera (accession number: FJ599761.1), query cover was at 98%. Eight parameters were used to measure the DNA polymorphism of thirty three homologous DNA sequences in this population. The results indicated that,  the number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotype (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (π), theta (per site) from Eta (Ø) were 5 (S), 5 (Eta), 2(h), 0,458(Hd), 2,292 (k), 0,314 x 10^-3 (π) and 0,169 x10^-3 (Ø), respectively (P < 0,05). Our analysis indicated that Rm of ITS4-5 genetic region in lotus populations did not appear (Rm = 0). Two methods (Tajima’s D test, Fu and Li’s D* and F* test) were used to execute neutrality test. The results showed that, the evolution of lotus population was balancing selection, sudden population contraction, in other words, rare alleles present at low frequency and there are very few individuals that show significant differences from other individuals in the population. Phylogenetic tree was built based on three methods, namely Neightbour -Joining, Maximum Likelihood và Maximum Parsimony (bootstrap = 1000) showed that, thirty three collected lotus samples closely linked and they were divided into two groups. Group I included 22 samples of pink lotus varieties and group II included 11 samples of white lotus varieties. Through this result, we found that the ability to distinguish between white and pink lotus varieties of N. nucifera was significant when using the ITS4-5 genetic region.