Twenty-nine Xanthium sibiricum Patrin germplasm resources from different ecological regions in Shandong province was analyzed for the genetic diversity of seed traits, and ISSR molecular markers in order to provide a basis for germpalsm research and genetic improvement. The results showed that the average kernel rate is 32.20%, and coefficient of variation is 26.66%, diversity index is 1.94. The average oil content is 30.03%, and coefficient of variation is 20.85%, diversity index is 1.94. The average hundred grain weight is 9.95g and coefficient of variation is 33%, diversity index is 1.96. 134 polymorphic loci were detected through 21 ISSR primers amplification with 6.38 loci per primer and the ratio of polymorphic loci is 98.53%. Different geographic and ecological group of Xanthium showed different genetic diversity. The highest genetic diversity was found in Luzhong mountain area, located in the junction of Jiaodong peninsula and inland, where gene exchanges take place easily. The lowest gene diversity was found in Lakeside lowland area, where it is blocked by Thai-Yi Mountain and Nansi Lake, the exchange of gene is limited. 29 Xanthium sibiricum resources in Shandong province plus one each from Hebei province and Shanxi province, clustering analysis, the results show that the Siberian cocklebur resources in Shandong province and neighboring provinces have certain genetic exchange, but has own characteristic, and has evolved its relative independent genetic resources system..