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Chinese fir (Cunninghamia lanceolata) is the most cultivated timber species in China, with a plantation area of 11 million ha. Due to its extensive geographical distribution, drought stress caused by the spatial and seasonal heterogeneity of precipitation has limited its survival and productivity. To facilitate the breeding of drought-tolerant clones and understand the inter-response mechanisms to drought stress, we screened two drought-tolerant (DT) clones and evaluated their differences in physiological and molecular response to drought. The results showed that the No. 228 clone (high-DT ability) had higher antioxidant enzyme abilities than the No. 026 clone (low-DT ability) under drought stress, e.g., peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), and catalase (CAT). Transcriptome analyses revealed that 6637 genes and 1168 genes were up-regulated in No. 228 and No. 026 under drought stress, respectively, when compared to the control (CK). The genes may participate in response to drought-stimulated signal transduction, water/oxygen-containing compound synthesis, photosynthesis, and transmembrane transport functions. Particularly, under drought stress, 14,213 up-regulated and differentially expressed genes (DEGs) were observed in the No. 228 clone compared with the No. 026 clone, and 4274 up-regulated genes were differentially expressed (15-fold difference). These significant DEGs were involved in plant hormone signal transduction, flavonoid biosynthesis, peroxisomes, and other key pathways related to drought. Interestingly, under drought stress, two Chitinases (ClCHIs) and four POD genes (ClPERs) were induced to express in No. 228, which was consistent with the higher antioxidant enzyme activities in No. 228. A heat map of 49 DEGs revealed that dehydrin family genes, ion binding/transmembrane proteins, auxin receptor proteins, and ethylene-responsive transcription factors were significantly up-regulated under drought stress. The results can enhance our understanding of drought tolerance mechanisms and provide a guideline for screening DT genes and breeding drought-tolerant Chinese fir clones. [ABSTRACT FROM AUTHOR]