Polyamines are ubiquitous and essential for cellular physiology, yet their metabolic pathways and functions remain only partially understood. Polyamine oxidases (PAO) are key to elucidating their physiological roles. In the methylotrophic yeast Ogataea parapolymorpha, we identified three putative PAO-encoding genes. Biochemical characterization showed that two of them function as PAOs, whereas the third has unknown substrate specificity. In contrast to previously studied yeasts, including Saccharomyces cerevisiae, which contain only a single PAO, O. parapolymorpha harbors multiple and functionally distinct PAOs. These findings highlight an unexpected diversification of polyamine catabolism in yeast and suggest previously unrecognized roles of PAOs in cellular physiology. © 2025 Elsevier B.V., All rights reserved.