Chloroplast DNA (cpDNA) in 84 Prunus L. accessions (interspecific hybrids and Prunus species) were analysed to confirm the maternal origin of the interspecific hybrids of Prunus and to establish genetic relationships among Prunus species. The polymerase chain reaction-restriction fragment length polymorphism (The PCR-RFLP) method, which included amplification of cpDNA regions with three universal primer pairs (K1K2, HK, DT) and subsequent digestion with three restriction enzymes (AluI, HinfI, TaqI), revealed 33 haplotypes for the 84 accessions studied. Fourteen from these cpDNA haplotypes were shared by two or more accessions, and 19 were unique. Accessions sharing the same haplotype have maternal relationships among them, which allowed identity confirmation of maternal progenitors of Prunus interspecific hybrids in these cases. Unweighted pair group method average (UPGMA) and minimum-length spanning tree (MST) analyses were performed based on shared common fragments and the number of mutational differences among the 33 haplotypes, respectively. The detected cpDNA polymorphisms enabled the analysis of genetic relationships among the studied Prunus accessions. Most of the recovered relationships are in agreement with current taxonomic hypotheses and artificial crosses.