Xiangru, with Mosla chinensis (Mc, 2n = 18) and its considered cultivar M. chinensis ‘Jiangxiangru’ (McJ, 2n = 18) as original plants, is an annual herb of the Lamiaceae family, and is widely used as medicinal and edible plant due to its spleen strengthening function. However, absence of genomic resource impedes in-depth research towards Xiangru. In this study, the morphological characteristics and volatile organic compounds (VOC) contents of Mc and McJ were analyzed, showing higher trichome density and monoterpenoid accumulation obtained in Mc, whereas McJ possessed higher biomass. We assembled high-quality Mc, McJ, and their adulterant Mosla soochowensis (2n = 18) genomes of 426.1, 408.8, and 412.8 Mb, respectively, containing the repeat sequences of 57.17%, 56.33%, and 55.83%. Comparative genomics analysis indicated Mosla radiated ~13.3 Mya, supporting McJ initially as a natural naturally formed resource. Five monoterpene synthase genes were identified through comparative transcriptome and were responsible for catalyzing production of diversified monoterpene skeleton, in which TPS1 mediated formation of γ-terpinene, accompanied by CYP71D179 and SDR2, leading to the final production of carvacrol and thymol. We further explored correlation between monoterpenoids biosynthesis and trichome development, indicating MIXTA and WIN1 jointly regulate both trichome formation and VOC accumulation by directly binding promoters of TPS1 and CYP71D179, respectively. Our study fills vacancy of genus Mosla genomes, improving the biosynthetic and regulatory mechanism of volatile compounds in aromatic Traditional Chinese Medicine, also offering novel targets for quality-directed breeding in Xiangru.

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