In this study, we characterized the gene expression profile in the roots of Populus ussuriensis at 0, 6, 12, 24, 48 and 120 h after the start of polyethylene glycol (PEG)-induced drought stress using PacBio single-molecule real-time sequencing (SMRT-seq) and Illumina RNA sequencing. Compared to the control, 2244 differentially expressed genes (DEGs) were identified, and many of these DEGs were associated with the signal transduction, antioxidant system, ion accumulation and drought-inducing proteins. Changes in certain physiological and biochemical indexes, such as antioxidant activity and the contents of Ca2+, proline, and total soluble sugars, were further confirmed in P. ussuriensis roots. Furthermore, most of the differentially expressed transcription factors were members of the AP2/ERF, C2H2, MYB, NAC, C2C2 and WRKY families. Additionally, based on PacBio SMRT-seq results, 5955 long non-coding RNAs and 700 alternative splicing events were identified. Our results provide a global view of the gene expression profile that contributes to drought resistance in P. ussuriensis and meaningful information for genetic engineering research in the future.