Abstract: In order to reveal the causes of low seed setting rate in the intersectional cross between herbaceous and tree peonies, hybridization was conducted between Paeonia lactiflora ‘Zhu Sha Pan’ and four tree peony cultivars ‘Feng Dan Bai’, ‘Dao Da Chen’, ‘High Noon’, and ‘Yao Huang’. Herbaceous peony cultivar ‘Kansas’ was taken as a control pollen parent. Pollen viabilities were tested in vitro. With aniline blue staining, the germinating rate of pollen grain and the growth process of pollen tubes in stigma were determined under fluorescent microscope. The results showed that the best pollination period of ‘Zhu Sha Pan’ was 4 d after blooming; pollen germination rate from high to low was ‘Kansas’, ‘Dao Da Chen’, ‘Feng Dan Bai’, ‘Yao Huang’, ‘High Noon’. Only pollen tubes from ‘Dao Da Chen’ and ‘Kansas’ could arrive at the ovary. In the growth process of pollen tubes, we also observed some unusual pollen tube morphology accompanied with a series of callose reactions. In conclusion, the incompatible reaction caused by pollen-stigma interaction could be one of the main barriers of intersectional crossing between Sect. Moutan and Sect. Paeonia.