Abstract: In this experiment, the ISSR system of Nymphaea nouchali was optimized by orthogonal design, and then the genetic diversity of Nymphaea nouchali (Hainan), Nymphaea nouchali (India), Nymphaea nouchali (Viet Nam) and 29 germplasm resources of Nymphaea nouchali were analyzed by ISSR molecular marker technology. The results showed that the optimal ISCR-PCR system was the total volume of 25μL (template DNA concentration 15 ng/μL,2×ES Taq Master Mix dosage 14μL, primer concentration 1.0μmol/L, ddH₂O complement). Ten primers with strong repeatability and good polymorphism were selected from100 ISSR primers using 8 germplasm materials such as Nymphaea nouchali as materials, and the annealing temperature was optimized. A total of 154 bands were amplified from 32 germplasm resources using the optimized system, with an average of 15.4bands per primer and 149 polymorphic bands (96.9%). The average shannon information index (Ⅰ) was 0.4582, the average allele number Na was 2.0000, the average effective allele number Ne was 1.5064, the average Nei’s gene diversity index H was 0.3001, and the genetic distance was 0.0355～0.7908. Based on the genetic distance, the clustering analysis diagram was constructed. The phylogenetic relationship between Nymphaea nouchali and 31 other Nymphaea species was explained, and the experimental materials could be divided into three types when the evolutionary distance was 0.2. The aim of this study was to clarify their genetic relationships. It laid the experimental foundation for the subsequent population genetic analysis of Nymphaeae nouchali, and provided reference for the identification of genetic relationships and the protection and utilization of planting resources.