As a plant-specific gene family, class III peroxidases (PODs) play an important role in plant growth, development, and stress responses. However, the POD gene family has not been systematically studied in Paeonia ostii. In this study, a total of 57 PoPOD genes were identified in the P. ostii genome. Subsequently, phylogenetic analysis and chromosome mapping revealed that PoPODs were classified into six subgroups and were unevenly distributed across five chromosomes. The gene structure and conserved motifs indicated the potential for functional divergence among the different subgroups. Meanwhile, four PoPODs were identified as tandem duplicated genes, with no evidence of segmental duplication. Using RNA-seq data from eight different tissues, multiple PoPODs exhibited enhanced expression in apical and adventitious roots (ARs). Next, RNA-seq data from AR development combined with trend analysis showed that PoPOD30/34/43/46/47/57 are implicated in the formation of ARs in tree peony. Through WGCNA based on RNA-seq, two key genes, PoPOD5/15, might be involved in heat tolerance via ABA and MeJA signaling. In addition, real-time quantitative PCR (qRT-PCR) analysis indicated that PoPOD23 may play an important role in flower senescence. These findings deepened our understanding of POD-mediated AR development, heat tolerance, and petal senescence in tree peony.