The chloroplastic DNA of 64 accessions of apple (Malus spp.) (54 Iranian genotypes, five commercial cultivars and five rootstocks) were analyzed to reveal their haplotypes by using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Approximately 4320 bp of the chloroplast genome was analyzed, by using two chloroplast universal primer pairs and two restriction enzymes (EcoRI and MseI). All the mutations detected were insertion-deletions been in the range between 10-40 bp. The combination of all the mutations resulted to six haplotypes (H1, H2, H3, H4, H5 and H6) in the studied apple accessions. It was defined that the Iranian apple rootstock 'Gami-Almasi' had a specific haplotype form all the other studied accessions. It was approved that cpDNA diversity data can be considered for phylogenetic studies in this plant and the polymorphism determined in cytoplasmic genome by PCR-RFLP method can help to understand the maternal inheritance in apple.