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The HSP20 family is the major member of heat shock proteins that are essential components involved in plant growth, development, and stress response, but little is known about tree peony (Paeonia suffruticosa). In this study, genome-wide analysis combining HSP20 gene family expression analysis of tree peony transcriptome was conducted, and the association between SNPs of HSP20s and flower organ number-related traits was analyzed. A total of 149 members were identified in the P. ostii genome, and divided into 10 subfamilies, most of which were classified into cytoplasm or nucleus. Interestingly, their protein sequences were highly conserved, mainly containing motif 1 or 3. In addition, a total of 38 deferentially expressed HSP20s were identified from transcriptome of flower buds with 5-carpels and polycarpels of P. ostii plants at three developmental stages, among which PoHSP89 and PoHSP133 were further cloned from 271 cultivars for association analysis. It indicated that seven or 19 pairs of associated combinations were obtained with the number of carpel, petal, stamen in PoHSP89 and PoHSP133, which could explain the phenotypic variation by 1.79% to 4.06%, and 1.92% to 12.37%, respectively. It will provide a valuable basis for clarifying the phylogenetic relationship to understand their biological function within the HSP20 gene family, and the identified candidate genes and their corresponding associated SNP loci would reveal the genetic basis on floral organ number variation and be useful for molecular marker-assisted breeding of tree peony in view to improving ornamental traits and yields.