The inheritance of chloroplast (cp) DNA was examined in F1 hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F1 offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.