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Background Interspecific hybridization has long been recognized as a pivotal process in plant evolution and speciation. It occurs fairly common in the genera of the subtribe Pyrinae. In Eriobotrya, a small tree genus of Pyrinae, E. prinoides var. daduheensis has been recognized as either a variety of E. prinoides, a natural hybrid between E. prinoides and E. japonica, or a variety of E. japonica. However, to date, there has been no convincing evidence on its status. Results Four nuclear genes and two chloroplast regions were sequenced in 89 individuals of these three Eriobotrya taxa from two locations where they coexist. A few fixed nucleotide substitutions or gaps were found in each of the investigated nuclear and chloroplast loci between E. japonica and E. prinoides. Of the 35 individuals of E. prinoides var. daduheensis, 33 showed nucleotide additivity of E. japonica and E. prinoides in at least one nuclear gene, and 10 of them harboured nucleotide additivity at all the four nuclear genes. Most haplotypes of E. prinoides var. daduheensis were also shared with those of E. japonica and E. prinoides. In the two chloroplast regions, 28 and 7 individuals were identical with E. japonica and E. prinoides, respectively. Conclusions Our study provides compelling evidence for a hybrid status for E. prinoides var. daduheensis. Most hybrid individuals are later-generation hybrids. Both E. japonica and E. prinoides can serve as female parent. Differential adaptation might maintain the species boundary of E. prinoides and E. japonica in the face of hybridization and potential introgression. [ABSTRACT FROM AUTHOR]