Abstract: Genetic diversity and genetic differentiation of Pseudotsuga gaussenii Flous were analyzed by using intersimple sequence repeat (ISSR) molecular technique.ISSR amplification was conducted with 10 primers for 76 individuals from 5 populations，and 69 polymorphic loci were detected from 76 loci，indicating high genetic variation at species level (PPB=90.79%，H=0.3361，I=0.4974) and at population level (PPB=56.58%-8.53%， H=0.2084-0.2681， I=02695-0.4076).A certain level of genetic differentiation occurred among populations (Gst=0.2366，Φst=26.88%).The program of genetic relationships among populations was constructed based on Nei’s gene distance.Two populations of Yangmingshan and Zhejiang clustered in a clade，then Anhui population and them clustered in a clade，and last Sanqingshan population clustered in a clade.In view of the genetic information available for .P.gaussenii. Flous，the number of .P.gaussenii. Flous has been dropping off.It is suggested that nature reserve should be founded in the distributing areas of P.gaussenii Flous to longtimely and effectively preserve the living space of P.gaussenii Flous.