Background: DNA methylation is an important biological form of epigenetic modification, playing key roles in plant development and environmental responses. Results: In this study, we examined single-base resolution methylomes of Populus under control and drought stress conditions using high-throughput bisulfite sequencing for the first time. Our data showed methylation levels of methylated cytosines, upstream 2kp, downstream 2kb, and repeatitive sequences significantly increased after drought treatment in Populus . Interestingly, methylation in 100 bp upstream of the transcriptional start site (TSS) repressed gene expression, while methylations in 100-2000bp upstream of TSS and within the gene body were positively associated with gene expression. Integrated with the transcriptomic data, we found that all cis -splicing genes were non-methylated, suggesting that DNA methylation may not associate with cis -splicing. However, our results showed that 80% of trans -splicing genes were methylated. Moreover, we found 1156 transcription factors (TFs) with reduced methylation and expression levels and 690 TFs with increased methylation and expression levels after drought treatment. These TFs may play important roles in Populus drought stress responses through the changes of DNA methylation. Conclusions: These findings may provide valuable new insight into our understanding of the interaction between gene expression and methylation of drought responses in Populus .