Cross-incompatibility is the major challenge in inter-sectional distant hybridization between tree peonies (Paeonia sect. Moutan) and herbaceous peonies (Paeonia sect. Paeonia). Most of the intersectional cross combinations rarely produce hybrid progenies or even seeds. However, a combination with Paeonia x lemoinei Rehd. (P. delavayi x P. suffruticosa, tree type) as the pollen parent and P. lactiflora (herbaceous) as the seed parent resulted in many progenies. This research focuses on cytogenetics of two highly compatible intersectional combinations, P. lactiflora 'Martha W.' x P. x lemoinei 'Golden Era' ('MW' x 'GE') and P. lactiflora 'Fen Yun Fei He' x P. x lemoinei 'L'Esperance' ('FYFH' x 'LE'). Karyotype analysis combined with genomic in situ hybridization (GISH) showed that the seed parents, 'MW' and 'FYFH', are both diploids (2n = 2x = 10, CC), while the pollen parents, 'GE' and 'LE', are tetraploid (2n = 4x = 20, AABB) and diploid (2n = 2x = 10, AB) respectively. Four progenies from 'MW' x 'GE', and another four progenies from 'FYFH' x 'LE', are all triploids (2n = 3x = 15, ABC). P. x lemoinei contributes two sets of chromosomes (AB) showing distinct signals in all tested triploids. The essential of a successful intersectional hybridization is to obtain 2x gametes from tree peony. P. x lemoinei has better compatibility in intersectional hybridization than other tree peony species, probably because of its higher frequency of 2x gamete (unreduced in diploid, or reduced in tetraploid) generation. Tetraploid tree peonies, such as 'GE', might produce abundant 2x gametes through normal meiosis, which is the possible reason for their better fertility and superior intersectional compatibility than diploid tree peonies. The mechanism of compatibility in intersectional hybridization is supposed to be a rebalance between the distant divergent parents, tree peonies and herbaceous peonies, triggered by ploidy increase of gametes from tree peonies. The discovery of tetraploid tree peony also provides valuable materials to study the mechanism of polyploidization in sect. Moutan, and to promote the distant hybridization breeding in Paeonia.