The mitochondrial genome of several poplar clones has been characterized by restriction analysis and hybridization with gene probes from Oenothera. The mitochondrial (mt) DNA of Populus has a complex restriction fragment pattern and its genome size was estimated to be about 450 kilobase pairs (kb). Restriction fragment length polymorphisms (RFLPs) could be detected only between, and not within, species of Populus and were used as genetic markers to follow mitochondrial inheritance. Location of the apocytochrome b (cob) gene on different Eco RI or Hin dIII fragments in Muhle Larsen (P. trichocarpa) and Androscoggin (P. maximowiczii ̸P. trichocarpa) has been used for analysis of mitochondrial inheritance. All hybrids investigated exhibit a fragment pattern identical to that of the female parent. Hybridization with other gene probes (coxIII/HindIII, coxIII/SalI, coxIII/Xho I, atp6/Bgl1, atp6/Hco RI, atp6/Hin dIII, nad1/Hin dIII, nad1/Sal I, nad1/Xba I) showed the same results as given by cob hybridization. Thus, mtDNA seems to be inherited maternally in Populus.