Cryopreservation is a vital technique for the long-term conservation of plant genetic resources outside their natural habitats, offering a reliable method to maintain pollen viability over extended periods. This study focuses on establishing a pollen cryobank for Nymphaea, a genus of ecological, ornamental, and economic significance. Nymphaea species contribute to freshwater ecosystems by enhancing water quality and supporting aquatic biodiversity. Their ornamental appeal makes them valuable in horticulture, yet habitat loss and climate change have led to the decline of many native populations, emphasizing the need for effective conservation strategies. The asynchronous flowering of Nymphaea species complicates hybridization efforts by limiting access to compatible pollen during peak flowering. Through cryopreservation, viable pollen can be stored year-round, preserving genetic diversity. This study developed and optimized cryopreservation protocols for seven Nymphaea species—N. pubescens, N. omrana, N. rosea, N. nouchali var. nouchali, N. nouchali var. versicolor, N. nouchali var. caerulea, N. micrantha, and N. malabarica. Key parameters such as optimal pollen collection time, in vitro germination media, and viability assessments were standardized. This protocol outlines the steps for collecting, drying, and cryopreserving pollen grains from Nymphaea species, as well as assessing their viability post-thaw. It provides detailed instructions on the preparation of pollen grains, the cryopreservation process, and the evaluation of pollen viability using in vitro germination techniques.