Genetic diversity and association analysis of Pueraria lobata resources is very useful to Pueraria lobata molecular breeding and fingerprinting construction. In this study, we carried out the genetic diversity and association analysis of 127 resources by using ten botanical characteristics and ISSR markers. The phenotyping results showed that Pueraria lobata have rich morphological diversity and all the ten botanical characteristics had great variances. 109 polymorphic bands of were amplified by 19 ISSR primers with an average of 5.73 loci per primer, the average value of Nei’s gene diversity is 0.2085, Shannon’s Information index is 0.3378 ,and the most distant GD is 0.46. The 127 resources were divided in two main groups by cluster analysis. PCA and STRUCTURE analysis result corresponding to the analysis by NTSYS, also divided 127 resources into two groups, there are three association signals by GLM association analysis and no association signals by MLM association analysis. The high level of genetic diversity was found in our resources, clustering analysis showed that there was little correlation between ISSR marker and geographical distribution, by comparing GLM and MLM association analysis result there wasn’t found any association signals.