The grapevine (Vitis spp.) is among the most extensively cultivated and economically important fruit crops in the world. The genus Vitis exhibits wide diversity, with American and Asian wild species possessing genes of interest that may be utilized for breeding climate-resilient varieties with high economic potential. Cryopreserving pollen offers a promising approach for conserving a part of species’ genetic diversity. Moreover, it plays a key role in breeding programs by helping to overcome spatial, seasonal, and physiological challenges in hybridization. In grape hybridization programs, cryopreserved pollens can be used to overcome the problem of breeding due to short flowering periods and asynchronous flowering between cultivated and wild grape species. The present chapter outlines a detailed procedure for cryostorage of pollen in three diverse Vitis species, including V. vinifera (Early Perlette Selection, Pusa Navrang, Pearl of Csaba, Perlette, Flame Seedless, Beauty Seedless, and Male Hybrid), V. champini (Dogridge and Salt Creek), and V. parviflora. It also details the correct stage of pollen collection and a simple desiccation protocol to harvest high quality and quantity of pollen for cryostorage. Pre- and postcryostorage pollen viability was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and validated through in vitro pollen germination on standardized media. Further, morphological differences in fresh and cryopreserved pollen were studied using scanning electron microscopy (SEM). The successful fruit and seed set achieved using cryopreserved pollen indicated that grape pollen can be effectively cryopreserved without any loss in viability during storage. This protocol will be valuable for breeders for efficient utilization of pollen, for grape conservation and improvement, thereby overcoming seasonal and temporal limitations in the availability of pollen.