Interspecific hybridization among different Luffa species facilitates the transfer or introgression of new genes for biotic and abiotic stress resistance or tolerance. However, this process often encounters several challenges, such as (a) mismatched flowering times due to different species’ sex forms and (b) temporal disparities in flowering periods. Cryopreservation is a handy tool for long-term storage of plant genetic resources (PGRs), and it can help to resolve the abovementioned problems. Pollen cryopreservation offers round-the-year availability of viable pollen grains in breeding programs irrespective of flowering habit and temporal variations. This chapter describes the detailed methodology of the Luffa pollen cryopreservation protocol, which holds promise for the long-term storage of pollen and its utilization in Luffa crop enhancement.