Fifteen interspecific hybrids of Serbian spruce (Picea omorika (Panc) Purkyne) and white spruce (Picea glauca (Moench) Voss) representing five separate crosses, including reciprocals, were used to demonstrate maternal inheritance of mitochondrial DNA. Total DNA was extracted from foliage samples of Serbian spruce (S), white spruce (W), and both S(♀) × W(♂) and W(♀) × S(♂) hybrids, digested and probed with one of two maize mitochondrial genes, ATPaseα or COXII. ATPaseα generated diagnostic Serbian and white spruce genotypes for all five enzymes tested, while COXII differentiated between the two species for four of five enzymes. Maternal inheritance was indicated in all hybrids for every diagnostic enzyme-probe combination. No paternal or nonparental bands were detected. A dilution experiment indicated that the Serbian and white spruce mitochondrial DNA restriction fragment length polymorphisms could be detected in as little as 60 and 500 ng of total DNA, respectively. It appears that the mechanism that controls the inheritance of mitochondria in Picea is still functional in wide interspecific crosses.