Forests face an escalating threat from the increasing frequency of extreme drought events driven by climate change. To address this challenge, it is crucial to understand how widely distributed species of economic or ecological importance may respond to drought stress. Here, we used RNA-sequencing to investigate transcriptome responses at increasing levels of water stress in white spruce (Picea glauca (Moench) Voss), distributed across North America. We began by generating an expanded transcriptome assembly emphasizing short-term drought stress at different developmental stages. We also analyzed differential gene expression at four time points over 22 days in a controlled drought stress experiment involving 2-year-old plants and three genetically unrelated clones. De novo transcriptome assembly and gene expression analysis revealed a total of 33,287 transcripts (18,934 annotated unique genes), with 4,425 unique drought-responsive genes. Many transcripts that had predicted functions associated with photosynthesis, cell wall organization, and water transport were down-regulated under drought conditions, while transcripts linked to abscisic acid response and defense response were up-regulated. Our study highlights a previously uncharacterized effect of drought stress on lipid metabolism genes in conifers and significant changes in the expression of several transcription factors, suggesting a regulatory response potentially linked to drought response or acclimation. Our research represents a fundamental step in unraveling the molecular mechanisms underlying short-term drought responses in white spruce seedlings. In addition, it provides a valuable source of new genetic data that could contribute to genetic selection strategies aimed at enhancing the drought resistance and resilience of white spruce to changing climates.