ABSTRACT Yuzu (Citrus junos Sieb. Ex Tanaka) is widely cultivated in Asia. However, a system for variety identification has not been developed yet. RNA-seq analysis was performed to identify variants among five yuzu samples including a seedless variety. However, only four variants were found, suggesting that the seedless trait might originate from bud mutations. To identify more variants, whole-genome resequencing was performed. A total of 140 reliable variants were screened. Based on these variants, 30 high-resolution melting (HRM) markers were developed. After genotyping 265 yuzu samples using the HRM markers, the yuzu samples were classified into 18 types. Three yuzu-like samples (B-37-1, B-UK2, and B-MD6-8) showing different fruit morphologies and off-type HRM peak patterns were assumed to originate from interspecific hybridisation. To identify the parent species of these yuzu-like samples, nucleotide sequences of a hypervariable chloroplast genomic region and a nuclear CitRWP gene responsible for nucellar embryony were obtained. Chloroplast genome sequence analysis indicated that the maternal parents of B-37-1 and B-MD6-8 were yuzu, whereas the B-UK2 sequence was identical to that of sweet orange. Moreover, the CitRWP sequences indicated that the male parents of B-37-1, B-UK2, and B-MD6-8 were mandarin, yuzu, and pummelo, respectively.