Taxus yunnanensis (Yew) is known for natural anticancer metabolite paclitaxel (Taxol) and its biosynthesis pathway in yew species still needs to be completely elucidated. In the current study, productions of paclitaxel and 10-DAB III from three different tissues (needle, branch, and root) of T. yunnanensis wild type (WT) and two new cultivars Zhongda-1 (Zd1) and Zhongda-2 (Zd2) were determined, and significant tissue differences in contents of the taxanes were observed among the three experimental lines. The much higher 10-DAB III and lower paclitaxel contents in needle of Zd2 when compared with that of Zd1 indicated the low conversion from 10-DAB III to paclitaxel in the needle of Zd2. In order to uncover the mechanisms of the tissue-specific biosynthesis of the taxanes, transcriptome analysis of cultivar Zd2 was conducted, and the previously reported transcriptome data of Zd1 and WT were used to perform a comparison. The enhancement of TDAT and T10βH side biosynthetic pathway in roots of Zd2 in early taxane synthesis might lead to the biosynthesis of other toxoids, while the preference of T13αH route in the needle and branch of Zd2 was mainly responsible for the tissue-specific reinforced biosynthesis of 10-DAB III and paclitaxel in Zd2. Different from Zd1, the tissue-specific pattern of paclitaxel biosynthesis genes in Zd2 was similar to WT. However, the lower transcript abundance of final steps genes (TBT, DBAT, BAPT, and DBTNBT) of the paclitaxel biosynthesis pathway in Zd2 than in Zd1 might further promote 10-DAB III accumulation in Zd2.