The Chloroplast DNA trnL- trnF region sequences from a natural hybrid species Meconopsis× cookei and its parents M. punicea and M. quintuplinervia were obtained by using direct sequencing method. The sequence length of trnL- trnF region is 960 bp for M. × cookei, 961 bp for M. punicea, and 957 bp for M. quintuplinervia. The sequences were aligned by the software Clustal X, and then the bases per locus were compared by using the software with manual method. The aligned sequence length is 964 bp, of which trnL intron is 512 bp, the 3′exon of trnL is 50 bp, trnL- trnF intergenic spacer ( IGS) is 361 bp, and the 5′end segment of trnF is 41 bp. Total 25 variable loci were detected from the aligned sequence, of which 21 (84% ) sites are same between M. × cookei and M. punicea, and only one (4%) is same between M. × cookei and M. quintuplinervia , the remaining three loci (12% ) are different among M. × cookei, M. punicea, and M. quintuplinervia. The results show that the cpDNA trnL- trnF region of the natural hybrid species M. ×cookei was inherited from its parent M. punicea. Therefore, according to the plastid inheritance law, our molecular evidences indicate that M. punicea is the mother of hybrid species M. × cookei and M. quintuplinervia is its father.