Abstract: High quality genome DNA of Pinus kesiya var. langbianensis was extracted from endosperm by the improved SDS method. After the DNA samples were completely digested by restriction enzymes of EcoRI and PstI and through preamplification and selective amplification, 24 pairs of primers out of 82 pairs with even band distribution, abundant polymorphism and high distinctiveness were selected. The distinct fingerprintings of Pinus kesiya var. langbianensis with high reproducibility were obtained through electrophoresis of the amplified product. The establishment of this AFLP mark system will be helpful to further study on Pinus kesiya var. langbianensis.