Agave americana L., commonly referred to as the century plant, is recognized for its remarkable antimicrobial properties and significant industrial potential. Its ability to thrive in arid environments and high yield makes it an attractive candidate for various applications, including fiber production and construction materials. To preserve its genetic diversity and protect biodiversity hotspots, the cryopreservation of A. americana pollen is essential. This study aimed to optimize a cryopreservation protocol that involved the collection of pollen using the bagging, assessment of pollen viability through the hanging drop technique and staining, and desiccation with silica gel-containing desiccators. The cryopreserved pollen was stored in cryogenic canisters. This protocol not only ensures the long-term conservation of the genetic resources of A. americana but also facilitates future research and educational opportunities. The findings underscore the importance of cryopreservation in safeguarding valuable plant species and promoting the sustainable utilization of their industrial and pharmaceutical potential.