Abstract: We used Populus tremula × P. tremuloides softwood cuttings as experimental material, and observed morphological and anatomical changes of the cuttings during the rooting process of adventitious roots. Solexa high-throughput sequencing was performed for the parts of cuttings where adventitious roots generated 6 days and 12 days after cutting (DAC) at transcriptional level. Anatomical observation showed that the induction stage of the adventitious root primordia was 6 days after cutting and the elongation stage of adventitious roots was 12 days after cutting. A total of 2 328 differentially expressed genes were obtained by the Solexa high-throughput sequencing. The significantly differential expression genes were classified after their functions annotation, and then the KEGG passway analysis was carried out. On this basis, the biochemical metabolic and signal transduction pathways of these differentially expressed genes were determined.