Hybridization across species has been widely utilized to improve agriculturally important traits in Rhododendron. These interspecific hybridizations are often inhibited by reproductive barriers at the prezygotic and postzygotic stages. Thus, in order to character the barriers and reveal the underlying regulation mechanism of interspecific hybrid incompatibility in Rhododendron, we compared the pollen germination and pollen tube growth, fruit growth, seed number and germination rate between intraspecific (Rhododendron ‘XXL’ × R. ‘XXL’) and interspecific (R. ‘XXL’ × R. delavayi) hybridizations. After pollination, the pollen germination rate and pollen tube growth rate of interspecific hybridization (Inter) were always higher than that of intraspecific hybridization (Intra) during the whole observation period. Conversely, the fruits and ovules of Intra grew faster than that of Inter after 20 DAP. The ratio of shrunken embryos of Inter was 73.27 ± 10.19 %, which was significantly higher than that of Intra with 28.41 ± 8.33 % at 40 DAP. Moreover, the fruit setting rate, the average number of seeds per fruit and seed germination rate of Intra were significantly higher than that of Inter at 150 DAP. These results indicated that the barriers of interspecific incompatibility between R. ‘XXL’ × R. delavayi could be mainly ascribed to post-fertilization isolation. Consequently, RNA-Seq analysis on ovaries at three developmental stages from two hybridizations indicated that plant hormone signal transduction, flavonoid biosynthesis, MAPK signaling pathway and plant-pathogen interaction pathways might play important roles in regulation of hybrid incompatibility between R. ‘XXL’ × R. delavayi. The results can provide a basis for the study of the hybrid incompatibility mechanism of Rhododendron, and provide useful information for the breeding and conservation of genetic resources of Rhododendron.