In our prior research on the fast-growing hybrid poplar Populus deltoides × Populus euramericana cv. ‘Nanlin895’, we identified key growth-related genes using the Populus trichocarpa genome as a reference. However, many discovered elements—such as newly annotated transcripts, promoters, regulatory sequences, and open chromatin regions—are poorly characterized, a situation often hindered by an incomplete genome assembly. These elements may critically influence shoot development through the regulation of quantitative traits. In this study, we validate the hybrid nature of ‘Nanlin895,’ showing that subgenome A (subA) and subB reflect the characteristics of P. deltoides and P. trichocarpa, respectively. We also illustrate the complementary expansions and contractions of gene families that contributed to allelic diversity. Subsequently, we conducted a detailed analysis of allele-specific gene expression, chromatin structure, and open chromatin regions in the shoot apical meristem, internodes 1–3, and internodes 4–5, which represent a gradient of shoot development. Analysis of asymmetrical expression identified 6279 genes, 3154 from subA and 3125 subB genes, that were differentially expressed during shoot development and revealed that subA and subB exhibit distinct expression patterns; for instance, genes associated with vascular functions are mainly expressed in subB, while those related to cell division are predominantly expressed in subA. By profiling topologically associating domains and open chromatin regions, we linked chromatin features to the regulation of key genes (e.g., CESAs, HB51, WOXs) that were expressed in different regions of the stem. These results reveal the genetic and three-dimensional chromatin basis of heterosis in ‘Nanlin895’, paving the way for molecular breeding strategies to enhance forestry and bioenergy traits.