Pollen cryopreservation is a vital technique for the long-term conservation of plant genetic resources, particularly for medicinally important species like Acorus calamus. This protocol details the systematic collection of viable pollen from healthy, mature plants during the flowering season, followed by ultra-low temperature storage in liquid nitrogen. Cryopreservation ensures the preservation of Acorus calamus genetic diversity, supporting future research, breeding programs, and conservation initiatives. By maintaining high pollen viability and germination rates post-thaw, this method serves as a crucial tool for biodiversity conservation and sustainable utilization of this valuable species.