Paeonia rockii is well-known for its distinctive large dark-purple spot at the white petal base and has been considered to be the main genetic source of spotted tree peony cultivars. In this study, the petal base and petal background of Paeonia ostii (pure white petals without any spot), P. rockii, and other three tree peony cultivars were sampled at four blooming stages from the small bell-like bud stage to the initial blooming stage. There is a distinct difference between the pigmentation processes of spots and petal backgrounds; the spot pigmentation was about 10 days earlier than the petal background. Moreover, the cyanin and peonidin type anthocyanin accumulation at the petal base mainly contributed to the petal spot formation. Then, we identified a C1 subgroup R2R3-MYB transcription factor, PrMYB5, predominantly transcribing at the petal base. This is extremely consistent with PrDFR and PrANS expression, the contents of anthocyanins, and spot formation. Furthermore, PrMYB5 could bind to and activate the promoter of PrDFR in yeast one-hybrid and dual-luciferase assays, which was further verified in overexpression of PrMYB5 in tobacco and PrMYB5-silenced petals of P. rockii by comparing the color change, anthocyanin contents, and gene expression. In summary, these results shed light on the mechanism of petal spot formation in P. rockii and speed up the molecular breeding process of tree peony cultivars with novel spot pigmentation patterns.