Chloroplast DNA (cpDNA) was purified from blue spruce (Picea pungens Engelm.) and white spruce [P. glauca (Moench) Voss], and was digested with several different restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated the cpDNA of both species. Intraspecific conservation of the RFLPs that differentiated each species was confirmed by examining trees from across the natural range of each species. Ten F1 hybrids were examined, and the cpDNA from each showed the banding pattern of the paternal species. Cloned Petunia cpDNA containing part of the rbcL gene hybridized to polymorphic bands, while a cloned maize mtDNA probe of the coxII gene failed to hybridize to any band.