Abstract: Medicinal materials in China differ in quality by different ecological types. Our research group found that there were two ecotypes of domestic Panax quinquefolium L. according to the characteristics of ginsenosides, inside versus outside Shanhaiguan. The genetic and ecological mechanisms of quality variation of Panax quinquefolium L. is unknown. Based on the genetic-chemical-ecological strategy, transcriptome and HPLC technology were used for comprehensive correlation analyses of transcriptomic data, ginsenoside content and environmental climate ecological factors. The transcriptomic results showed that key genes of ginsenoside biosynthesis, such as HMGR, AS and FPS, were significantly down-regulated in the inside Shanhaiguan ecotype. HPLC results showed that the quality of outside Shanhaiguan ecotype Panax quinquefolium L. was higher than that of the inside ecotype, with the content of ginsenosides in outside Panax quinquefolium L. was higher than that of inside ecotype except Rb₂. Correlation analyses revealed that content of Panax quinquefolium L. ginsenoside is positively related to the expression levels of ginsenoside biosynthesis key genes (MK, HMGS, HMGR, and AS), and negatively related to the expression of glycosyl transferase (GT). The content of ginsenosides is negative related with climate factors, such as temperature, sunshine, and is positively related with moisture in both ecological environments. This study has provided a new mechanistic insight into the quality variations of two ecotypes for Panax quinquefolium L. and established a scientific basis for studying the ecological factors for the quality of traditional Chinese medicine.