Widely targeted metabolomics was used to study the difference of metabolite contents in the needles of Taxus wallichiana var. mairei and T. wallichiana var. mairei cv. ‘Jinxishan’. The results showed that: (1) a total of 689 metabolites and their integral quantitative values were found, including 326 primary metabolites, 334 secondary metabolites and 29 other components. (2) The results of quantitative analysis showed 71 metabolite contents were significantly difference. Most of differential metabolites between T. wallichiana var. mairei and T. wallichiana var. mairei cv. ‘Jinxishan’ were significant enriched in primary metabolic pathways, such as glycometabolism and lipid biosynthesis pathways, as well as secondary metabolic pathways such as flavonoids biosynthesis pathways. (3) A large number of metabolites in the amino acid and flavonoid biosynthesis pathways were significantly accumulated in T. wallichiana var. mairei cv. ‘Jinxishan’, while the differential metabolites in sugar metabolism, lipid synthesis and TCA cycle pathways were mainly accumulated in T. wallichiana var. mairei. The study considered that the increase of flavonoid content was attributed to the coregulation of phenylalanine and tyrosine metabolic pathways, which improved the adaptability of T. wallichiana var. mairei cv. ‘Jinxishan’ to the environment. At the same time, the energy was supplied by synthesizing large amounts of amino acids to make up for the lack of energy, caused by the decrease of metabolite contents in glycometabolism, lipid biosynt hesis and TCA cycle pathways.