【Objective】Nymphaea spp. (waterlily) are important aquatic flowers. The rapid development of the waterlily industry has led to challenges in identifying genetic backgrounds of newly introduced germplasms in a timely manner due to inadequate introduction management practices. This has resulted in issues such as mislabeling of seedlings and unclear parentage, hampering the effective utilization and innovation of waterlily germplasm resources. This study focuses on developing genome-wide SSR markers for conducting phylogenetic and genetic diversity analyses of waterlily species. These markers are intended to serve as a theoretical foundation for conserving and breeding waterlily germplasm resources.【Method】Based on the published complete genome sequence of waterlily, the SSR loci in 14 chromosome genes were analyzed using the micro satellite identification (MISA) tool. Subsequently, 150 pairs of SSR primers were designed with the assistance of Primer 3.0 software. Five native germplasms were chosen for PCR amplification utilizing the 150 primer pairs. Following PCR, SSR primers demonstrating high polymorphism were identified through agarose and denaturing polyacrylamide gel electrophoresis. The selected SSR primers were synthesized with fluorescent primers (FAM and HEX) before amplifying 147 waterlily samples via PCR. The resulting products were then assessed using capillary electrophoresis, and the raw data were analyzed using GeneMarker software. Fragment sizes at each allele site for every sample were compiled into a 0/1 matrix. Genetic diversity indices, cluster analysis, and principal component analysis were computed using Popgenen and NTSYS software toos.【Result】11 pairs of SSR primers exhibiting distinct bands and high polymorphism were carefully chosen following the analysis of agarose gel and polyacrylamide gel electrophoresis results. These selected primers were employed to evaluate the genetic diversity among 147 waterlilies. Capillary electrophoresis revealed the presence of 307 alleles. The polymorphism index (PIC) ranged from 0.46 to 0.60, average at 0.53. The effective allele number (N_e) rangedfrom 1.042 8 to 1.117 5, with an average of 1.071 8. Nei’s gene diversity index (H) ranged from 0.038 0 to 0.086 2, averaging at 0.056 2. The Shannon diversity information index (I) ranged from 0.085 6 to 0.163 8, averaging at 0.114 4. Cluster analysis indicated genetic similarity coefficients ranging from 0.781 8 to 0.993 5 among the 147 waterlilies, with an average coefficiency of 0.899 2. These waterlilies were classified into six groups based on a genetic similarity coefficient was 0.879 0. Principal coordinates analysis (PCoA) displayed results closely aligned with traditional morphological classification for the 147 waterlilies. The first, second and third principal coordinates accounted for 16.54%, 8.35% and 5.43% of the total genetic variation, respectively, comprising 30.32% altogether. The first coordinate was correlated with aroma formation and stamen development, the second with flowering time and environmental adaptability, and the third with flower types.【Conclusion】11 pairs of SSR primers displaying substantial polymorphism were chosen, demonstrating efficacy in distinguishing the genetic relationships among 147 waterlilies. These waterlilies were systematically categorized into six primary branches, with classification outcomes mirroring traditional morphology categorizations. The selected 11 pairs of SSR primers have potential utility in analyzing genetic diversity and identifying waterlily species. The SSR markers findings stand poised to offer a robust scientific foundation for germplasm collection, preservation, innovation, and the development of waterlily species.