The hybrid origin of Ilex ${ imes}$ wandoensis C. F. Mill. & M. Kim was studied by polymerase chain reaction (PCR) using taxa-specific primers, restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer 2 (ITS2) regions, and sequence analysis of PCR amplicons of the ITS region. Five accessions of I. integra Thunb., three accessions of I. cornuta Lindl. & Paxton and 22 accessions of I. ${ imes}$ wandoensis, tentatively characterized based on morphological characters, were selected and subjected to PCR analysis. Amplified products of the selected accessions were sequenced. Significant variations were observed in leaf morphology of I. ${ imes}$ wandoensis accessions based on the presence of single or multiple teeth and cuneate or rounded base of the leaf blade. Based on the PCR amplifications, sequence and RFLP analysis using HhaI and HaeIII restriction enzymes, the hybrid origin of I. ${ imes}$ wandoensis was confirmed. Some accessions appeared to be introgressant and either crossed back to I. integra or to I. cornuta based on the RFLP polymorphisms. Analysis of the ITS2 region of the nuclear ribosomal DNA (nrDNA) using primers specific to certain taxon or taxa, and RFLP analysis of PCR amplicons proved to be useful tools to study the nature of interspecific hybridization and introgressions in complement with morphological characters.