UNLABELLED: Polyamine oxidases (PAOs) are enzymes degrading the polyamine molecules and have important roles in plant growth, development and in stress tolerance. Despite their significance, their genomic organization and functional roles in Sorghum bicolor, a drought-tolerant staple crop, remain largely unexplored. In this study, a comprehensive comparative genomics analysis was conducted and identified six PAO genes in sorghum phylogenetically clustered into four clades, with sorghum exhibiting lineage-specific expansion via segmental and tandem duplications. Structural modelling identified conserved FAD-dependent oxidase cores across all SbPAO proteins and identified a novel motif (GLRLYRTSGDNSVLYDHDLEDYALYDYEGAQVPRETVLK) unique to sorghum PAOs, potentially linked to flavin-dependent oxidoreductase activity. SbPAO5 and SbPAO6 exhibited the most elaborated fold in three-dimentional modeling. SbPAO4 and SbPAO5 possess peroxisomal targeting signals (PTS1). These structural and targeting divergences collectively suggest subfunctionalization within the SbPAO family. Collinearity analysis highlighted strong syntenic conservation with rice and maize suggesting evolutionary and functional conservation of PAOs in grasses. Promoter sequence analysis revealed presence of several stress and hormones responsive elements, aligning with tissue- and genotype-specific expression patterns under drought. In the tolerant genotype Dorado, SbPAO4–6 were dynamically upregulated in leaves and grains, correlating with spermidine accumulation and enhanced stress resilience. Elevated spermidine level in sensitive genotype Giza 15 suggested back conversion of spermine to spermidine by upregulation of SbPAO5. Co-expression analysis linked SbPAO5 and SbPAO6 to stress signalling and metabolic hubs implicating their roles in integrated stress adaptation. These findings establish a foundation for genomic organization and evolutionary relationships of PAO genes in sorghum and prioritizes SbPAO5 and SbPAO6 as candidates for stress-resistant breeding and enhanced production in sorghum.SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12125-4.