The genus Thuja is ideal for investigating the genetic basis of the East Asia-North America disjunction. The biogeographical background of the genus is debatable and an adaptive strategy is lacking. Through the analysis and mining of comparative transcriptomes, species differentiation and positively selected genes (PSGs) were identified to provide information for understanding the environmental adaptation strategies of the genus Thuja. De novo assembly yielded 44,397-74,252 unigenes of the five Thuja species with contig N-50 length ranging from 1,559 to 1,724 bp. Annotations revealed a similar distribution of functional categories among them. Based on the phylogenetic trees constructed using the transcriptome data, T. sutchuenensis was divided first, followed by T. plicata and T. occidentalis. The final differentiation of T. koraiensis and T. standishii formed a clade. Enrichment analysis indicated that the PSGs of the North American Thuja species were involved in plant hormone signal transduction and carbon fixation of photosynthetic organisms pathways. The PSGs of East Asian Thuja were related to phenolic, alkaloid, and terpenoid synthesis, important stress-resistant genes and could increase plant resistance to external environmental stresses. This study discovered numerous aroma synthetic-related PSGs including terpene synthase (TPS) genes and lipid phosphate phosphatase 2 (LPP2), associated with the synthetic aroma of T. sutchuenensis. Physiological indicators, such as the contents of soluble sugars, total chlorophyll, total phenolics, and total flavonoids were determined, which are consistent with the PSGs enrichment pathways associated with adaptive strategies in the five Thuja species. The results of this study provide an important basis for future studies on conservation genetics.