Calcium-dependent protein kinases (CDPKs) function as essential sensors and transducers of intracellular calcium signals, playing crucial regulatory roles in various physiological processes. Nevertheless, studies on CDPK genes in Paeonia ostii remain limited. In this study, 16 PoCDPK genes containing a complete Pkinase domain and four EF-hand domains, distributed across five chromosomes, were identified in P. ostii. Phylogenetic analysis classified these genes into four distinct groups (I–IV). Promoter cis-element analysis suggested a potential involvement of PoCDPK genes in plant growth and development, phytohormone signaling, light, circadian, and stress responses. Transcription levels of PoCDPKs were highest at the early stage of root primordium formation (Gmfq) among the four stages examined. Expression analysis of the 14 PoCDPK genes detected in the transcriptome revealed that nine PoCDPKs were highly expressed in flowers, two in stems, PoCDPK10 in leaves, PoCDPK13 in roots, and PoCDPK1 in roots, stems, and leaves. Moreover, PoCDPK3, PoCDPK12, and PoCDPK13 from Group I, as well as PoCDPK2 and PoCDPK8 from Group III, were highly expressed at 5 d of rooting induction, suggesting their potential involvement in adventitious rooting. These findings provide a comprehensive reference for the potential functions of the CDPK family in the adventitious rooting of tree peony.