Abstract: The cDNA amplified fragment length polymorphism (cDNA-AFLP) system for Populus euphratica was established after optimizing several key factors, which may affect cDNA-AFLP analysis. The results indicated that optimized CTAB method could be suit for extracting ideal RNA from P. euphratica; the ds-cDNA could be digested completely by the combination of EcoRⅠ and Mse Ⅰ for 4.5 hrs; the reducible result can be obtained when the pre-amplification products were diluted to 10 times for selective amplification template; the amplified products resulted from this reaction system were determined by silver-staining, and the fragments were visualized clearly and stably; detected by RT-PCR, the optimized differential display cDNA-AFLP technique can accurately reflect the true differences of gene expressions among plants. In addition, 42 pairs of primer arrays, producing repeatable and prolific bands, were successfully selected by this improved method. This investigation establishes a cDNA-AFLP system for P. euphratica, which builds a foundation for further study on stress-response genes in P. euphratica.