Whole genome sequencing analysis reveals strong reproductive isolation between two hybridizing Rhododendron species in subgenus Tsutsusi

•Whole genome sequencing analysis reveal the first case of natural hybridization in <i>Rhododendron</i> subgenus <i>Tsutsusi</i> in China.•Most examined <i>Rhododendron</i> <i>strigosum</i> plants (putatively hybrid origin) were F1s, indicating nearly complete reproductive isolation between the two parental species.•We further revealed the rare formation of F2s after detecting seedlings from seeds of ex-situ conserved <i>Rhododendron</i> <i>strigosum</i> under natural pollination.•<i>Rhododendron</i> <i>hypoblematosum</i> had both the highest number of homozygous deleterious mutations and the highest ratio of homozygous deleterious mutations to synonymous mutations, exceeding that of the F1s.