Sequence and restriction site analyses of the paternally inherited chloroplast rbcL gene and maternally inherited mitochondrial nad1 fragments from the same set of populations and individuals were used to investigate cytoplasmic composition and population establishment of Pinus densata, a diploid pine that originated through hybridization between P. tabuliformis and P. yunnanensis. Two variable sites and three chlorotypes (TT, TC and GC) were detected on the rbcL gene of the three pines. P. densata harboured the three chlorotypes, two of which (TT, GC) were characteristic of the parental species, respectively. The third chlorotype (TC) was distributed extensively in seven of the 10 P. densata populations analysed, and might represent a mutation type or have been derived from an extinct parent. The distribution of chlorotypes, together with that of mitotypes, indicated that significant founder effect and backcross happened during the population establishment of the hybrid pine. P. tabuliformis and P. yunnanensis had acted as both mother and father donors, i.e. bi-directional gene flow existed between the two parental species in the past. Population differentiation of P. densata is high, as detected from the cytoplasmic genomes: GST = 0.533 for cpDNA and GST = 0.905 for mtDNA. The differences in cytoplasmic composition among the hybrid populations suggest that the local populations have undergone different evolutionary histories.