Abstract: Background and Objective: Populus species are one of the world’s most important groups of forest trees, but distinguishing between cultivars and hybrids is a little difficult due to widespread interspecific hybridization and introgression and the high level intraspecific morphological variation. The objective of this study was to understand the genetic variation and heterozygosity of 29 Populus species from the Jianghan Plain of China and provided scientific guidance for the introduction of superior poplar varieties in Hubei province of China. Materials and Methods: The DNA was isolated from leaves of 29 poplar clones, digested by MseI and EcoRI endonucleases and subsequently used Amplified Fragment Length Polymorphisms (AFLP) analysis. The AFLP data was analyzed by GeneMapper software to display the fragment sizes as electropherograms and binary data. Fingerprint was constructed according to the AFLP analysis. Genetic similarity coefficients were calculated using Numeric Taxonomic System (NTSYS) PC version software 2.11 and the phylogenetic tree was constructed by cluster analysis using the UPGMA. Results: A total of 959 fragments were amplified, 806 of which were polymorphic with an average polymorphism percentage of 84.67%. Genetic similarity coefficients of the 29 Populus species ranged from 0.6111-0.8478 with an average of 0.7217. The highest average genetic similarity coefficient (0.7549) was found between "Danhongyang" with other Populus species and the lowest (0.6532) was found between "6204" with other Populus species. According to the genetic similarity coefficient of 0.7117, the 29 Populus species were divided into four categories by UPGMA cluster analysis. Finally, we built DNA fingerprint of the 29 Populus genotypes. Conclusion: It was concluded that 29 Populus species shared high genetic similarity and low genetic diversity. "107", "6204" and "6102" were three unique species and worth further utilizing in hybridization.