Pyroleae (Ericaceae) consist of four genera, all of which are distributed widely in temperate coniferous or sometimes deciduous forests of the Northern Hemisphere. To investigate the phylogenetic relationships among these genera and to explore the evolution of the characteristics of the subfamily, we conducted maximum parsimony and Bayesian analyses with nrDNA ITS and three cpDNA intergenic spacers (atpB-rbcL, trnS-trnG and trnL-trnF). The results from cpDNA and combined cpDNA + ITS data sets strongly support the monophyly of Pyroleae as well as a sister relationship between Pyrola and Moneses-Chimaphila, with Orthilia as the basal lineage. The sister-group relationship between Moneses and Chimaphila is supported by a set of synapomorphies, e.g., single flower, colpate pollen, five bundles in the style, straight fruiting pedicel orientation, complete capsule dehiscence, and the basic chromosome number, x = 13. The Moneses-Chimaphila-Pyrola clade is supported by at least one homologous character of pollen in tetrads. Conflicts associated with the phylogenetic position of Orthilia may imply a hybrid origin for it, and therefore further study is needed.