Vigna vexillata (L.) A. Rich., known as tuber cowpea, Zombi pea, and wild cowpea, is a legume that thrives in diverse climates and shows resilience against bruchid and abiotic stress. V. vexillate is a close relative of the cultivated cowpea, and it is cultivated for its storage roots, protein-rich seeds, forage, and erosion control capabilities. It grows in both protected and partially disturbed habitats in hilly and sub-hilly regions of peninsular India and the Himalayas. The cryopreservation of pollen grains is a vital technique for conserving important alleles and making genetic resources readily available for breeding programs. This method overcomes the challenge of asynchronous flowering, facilitating the creation of hybrids with pest and disease resistance, high yield, and desirable qualities. To advance wide hybridization studies, pollen cryopreservation was evaluated across 12 accessions of V. vexillata. Fresh pollen viability was recorded between 85.72% and 93.56%. This study assessed pollen viability under different storage conditions (25, 4, -20, and -196 °C) and various durations (1, 3, 5, 7, 9, and 24 hours; 1, 2 weeks; and 1 month). Optimal long-term cryopreservation was achieved with a pollen moisture content of 10-12% following a 5-minute desiccation period in a laminar airflow chamber. A significant negative correlation was observed between pollen viability, storage duration, and temperature (25, 4, and -20 °C) across all accessions. Pollen stored at -196 °C demonstrated significantly higher viability, ranging from 81.14% to 93.07%, compared to other storage conditions. The established cryopreservation protocol successfully maintained pollen viability for up to one month and enabled effective pollination, fruit development, and seed set in crosses using cryopreserved pollen.