Abstract: The morphyological characters of five kinds of Boletus in Yunnan were observed,and their r DNA ITS and IGS1 sequences of were measured,analysed and contrasted with the sequences of Boletus having been logged in Gen Bank. Phylogenetic tree was constructed by the obtained molecular data. The results showed that the total length of all the tested material ITS and IGS1 was respectively within the scope of 739~787bp and537~538bp. the content of GC was respectively within the scope of 48.31%~51.01% and 48.42%~52.89%,the range of genetic distances was respectively between 0.017~0.248 and 0.002~0.225. The datas showed that there was certain genetic differences between five kinds of Boletus. The five kinds of Boletus could be divided into two groups by their ITS and IGS1 sequence clustering analysis,YX1-2 and QJ3-4 formed in one group,CX2-10,QJ3-5 and YX1-3 formed into another group,two analytical methods confirm each other,and five samples from different regions of Yunnan were identified to genus level and even species based on the ITS sequence clustering analysis,it showed that combining the ITS with IGS1 sequences could effectively identify the genetic relationship of Boletus.