Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.