The four-subfamily subdivision of Rosaceae has been recently replaced by a three-subfamily scheme. The re-circumscribed Rosoideae lacks a solid and well-resolved phylogeny on which a classification can be based. In this study, we sampled 56 genera presumably belonging to Rosoideae and 10 genera belonging to other subfamilies or families and used 12 chloroplast regions (matK, rbcL, trnL, trnL–F, ndhF, ycf1, trnC–ycf6, trnS–G, trnS, psbA–trnH, rpoC1 and trnS–ycf9) to reconstruct their phylogeny. Our results confirmed (1) the exclusion of Rhodotypos and Kerria from Rosoideae and their inclusion in the subfamily Amygdaloideae and (2) the exclusion of Chamaebatia, Cercocarpus, Dryas and Purshia (including Cowania) from Rosoideae and their inclusion in Dryadoideae, the sister subfamily of Rosoideae. Within Rosoideae, there are six strongly supported lineages that correspond to six tribes: Ulmarieae, Colurieae, Rubeae, Roseae, Agrimonieae and Potentilleae. We dated the divergence of Rosoideae back to approximately 69.77 million years ago (Mya; 95% HPD = 61.28–78.33 Mya) and that of the tribes within Rosoideae to from 10.42 to 40.02 million years ago (Mya; 95% HPD = 4.73–59.08 Mya). The subfamily is probably of North American and Asian origin and thrives in the northern hemisphere, especially in Asia. After re-circumscriptions of several genera, there are 36 genera recognized in Rosoideae.