<p><i>Nymphaea nouchali</i> Burm f is a perennial aquatic flowering herb native to Southeast Asia, is valued for horticultural and medicinal uses. As an underexplored herb, understanding their genetic diversity is crucial for conservation and breeding programs. Hence, the study intended to assess the genetic variability among 61 <i>N. nouchali</i> accessions collected from various districts of Kerala, India, using inter-simple sequence repeat (ISSR) markers. Of the 50 primers tested, 21 produced scorable banding patterns, yielding 190 ISSR fragments averaging 9.05 bands. On average, 74.74% of the bands were polymorphic, with primer UBC 815 exhibiting the highest polymorphism (93.75%). Polymorphic information content (PIC) values ranged from 0.30 to 0.405, indicating polymorphism and genetic variation among accessions. The effective multiplex ratio (EMR) ranged from 4.23 to 8.30 (averaging 6.27), while the marker index (MI) ranged from 1.301 to 3.223 (averaging 2.139), with a positive correlation between them. The mean Shannon indices (0.2963) and Nei’s gene diversity (0.19) indicated a moderate genetic variation within the accessions. The dendrogram constructed using the neighbor-joining method with 100 bootstrap values classified the accessions into three clusters, showing that genetic similarity was not always correlated with geographical proximity. STRUCTURE analysis indicates the presence of three subpopulations based on Bayesian clustering. This suggests complex gene flow and historical germplasm exchange. In the present study, only moderate genetic diversity was observed due to various environmental factors. In brief, the investigation offers key insights into the efficiency of ISSR markers and the genetic diversity of species, underscoring the need for management and conservation.</p>