Peony is a famous ornamental and medicinal plant in China, and peony hybrid breeding is an important means of germplasm innovation. However, research on the genome of this species is limited, thereby hindering the genetic and breeding research on peony. In the present study, simple sequence repeat (SSR) locus analysis was performed on expressed sequence tags obtained by the transcriptome sequencing of Paeonia using Microsatellite software. Primers with polymorphism were obtained via polymerase chain reaction amplification and electrophoresis. As a result, a total of 86,195 unigenes were obtained by assembling the transcriptome data of Paeonia. Functional annotations were obtained in seven functional databases including 49,172 (Non-Redundant Protein Sequence Database: 57.05%), 38,352 (Nucleotide Sequence Database: 44.49%), 36,477 (Swiss Prot: 42.32%), 38,905 (Clusters of Orthologous Groups for Eukaryotic Complete Genomes: 45.14%), 37,993 (Kyoto Encyclopedia of Genes and Genomes: 44.08%), 26,832 (Gene Ontology: 31.13%) and 37,758 (Pfam: 43.81%) unigenes. Meanwhile, 21,998 SSR loci were distributed in 17,567 unigenes containing SSR sequences, and the SSR distribution frequency was 25.52%, with an average of one SSR sequence per 4.66 kb. Mononucleotide, dinucleotide, and trinucleotide were the main repeat types, accounting for 55.74%, 25.58%, and 13.21% of the total repeat times, respectively. Forty-five pairs of the 100 pairs of primers selected randomly could amplify clear polymorphic bands. The polymorphic primers of these 45 pairs were used to cluster and analyze 16 species of peony. The new SSR molecular markers can be useful for the study of genetic diversity and marker-assisted breeding of peony.