Chloroplast markers are powerful tools for research into relationships among grapevine (Vitis vinifera L. subsp. vinifera) cultivars. However, the high number of regions common to the chloroplast and mitochondrion genomes described in grapevine could interfere with correct phylogeny reconstruction. In this study, we established a unique chloroplast marker (UCM) set and investigated the chloroplast haplotype diversity of 17 grapevine cultivars and seven Asian Vitis species. Sequencing of four UCMs revealed four haplotype groups in grapevine based on three nucleotide substitutions, three simple sequence repeats, and one 54 nucleotide (nt) deletion in the trnC-petN region. The constructed molecular-variance parsimony and statistical parsimony networks indicated that two independent maternal lineages of grapevine cultivars originated from a core group of Asian Vitis species. The presence and absence of a newly discovered 54 nt deletion corresponded to the two maternal lineages: deletion (D) and non-deletion (ND) lineages. The D lineage consisted of three haplotypes, represented by 'Furmint', 'Gouais blanc', and 'Sauvignon blanc'. Ancient noble cultivars, such as 'Pinot noir', 'Riesling', and 'Afus ali', belong to a single haplotype group (ND) in which the deletion was absent. Our results provide compelling evidence for the dual maternal origin of grapevine, and suggest the occurrence of an ancient hybridization event.