For red-flowered cultivars of tree peony(Paeonia suffruticosa), anthocyanin content is a critical factor determining the different petal pigmentations. Glutathione S-transferases (GSTs) are ubiquitous and multifunctional conjugating proteins that may be responsible for the transport of anthocyanin pigments from the cytoplasm to vacuole. The underlying function of the GST family in tree peony, however, remains unclear. In this study, we systematically isolated and identified a total of 54 putative full-length PsGST genes through a combination of bioinformatics approaches from transcriptome databases. Intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into 10 of the 14 known classes of plant GSTs. The phylogenetic relationships, evolutionary characteristics, protein domain, and motif organization were clearly conserved among the different phylogenetic subclasses. The results of the RNA-seq and quantitative real-time polymerase chain reaction experiments exhibited extensive variation in gene expression profiles among different developmental stages and varieties. Furthermore, the phylogenetic relationships, expression profiles, protein interactions, weighted gene co-expression network analysis, and correlation analysis results suggested that PsGSTF3 (Unigene 0064200) is a candidate participant in anthocyanin transport and the promotion of pigment accumulation, exhibiting a strong positive correlation with anthocyanin content among different tissues (r = 0.908⁎⁎) and an increasing rate of anthocyanin content during the flower developmental process (r = 0.961*). These results furthered our understanding of the transport and accumulation functions of the GST family as well as the enhancement of tree peony breeding through molecular biology techniques.