We used restriction fragment analysis of chloroplast, nuclear, and mitochondrial DNA to study phylogeny in the genus Pinus. Total genomic DNA of 18 to 19 pine species that spanned 14 of the 15 subsections in the genus was cut with 8 restriction enzymes, blotted, and then probed with up to 17 cloned DNA fragments-which were mostly from the chloroplast genome of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). A total of 116 shared characters, the majority representing single point mutations, were subjected to Wagner and Dollo parsimony analyses, coupled with bootstrapping and construction of consensus trees. The hard (subgenus Pinus) and soft pines (subgenus Strobus) were distinct. The soft pines in section Parrya, represented by P. longaeva, edulis, monophylla, and gerardiana, were the group closest to the hypothesized root of the genus. They were also more diverse and more closely related to the hard pines than were their descendents in section Strobus, represented by P. koraiensis, albicaulis, griffithii, and lambertiana, all of which were remarkably similar. Except for a strong clade involving P. canariensis and pinea (section Ternatae), the hard pines were weakly differentiated. The high similarity within the most speciose groups of pines (sections Strobus and Pinus) suggests that the bulk of the genus radiated relatively recently. In contrast to a recent classification, P. leiophylla was not associated with section Ternatae; instead, it appears to belong in section Pinus, and showed a high similarity to P. taeda of subsection Australes. Subsection Oocarpae, represented by P. oocarpa and radiata, appears to be a natural group, and is related to subsection Contortae, represented by P. contorta. More extensive restriction fragment studies will yield many new insights into evolution in the genus. Other methods, however, such as DNA sequencing or fine structure analysis of restriction site mutations, are likely to be necessary for rooting pine phylogenies with respect to other coniferous genera, and for estimating divergence times.