The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.