Abstract: In order to analyze the genetic diversity of Macleaya cordata germplasm resources in 13 provinces of China, the genetic relationship and genetic differentiation were clarified. The genetic diversity of 16 germplasm resources of Macleaya cordata was analyzed by capillary electrophoresis with SSR fluorescent markers. The polymorphic information content (PIC) values of 16 Macleaya cordata germplasm resources ranged from 0.375 to 0.687, and the genetic similarity coefficient ranged from 0.0714 to 0.7142. The cluster analysis results showed that: the 16 germplasm resources of Macleaya cordata could be divided into three major categories at the similarity coefficient of 0.3. There were 7 germplasm resources in the first major category, 1 germplasm resource in the second major category and 8 germplasm resources in the third major category. In addition, the DNA fingerprint database of 16 Macleaya cordata germplasm resources was constructed, and the corresponding fingerprints were developed. The results could provide available molecular markers and reference for the classification and identification, genetic diversity analysis and variety breeding of Macleaya cordata germplasm resources.