Atractylodes lancea, known as Cangzhu in Chinese , has been widely used in the traditional Chinese medicine (TCM). The colour of secretory cavity (SC) at Atractylodis Rhizoma (AR) differs greatly among geographic origins and cinnabar-like red SCs of AR is considered of high quality in TCM. However, chemical basis underlying this colour variation across natural accessions and within plant tissues are largely unknown, impeding implications of efficacy of cinnabar-like spots for Cangzhu in TCM. Here, we carried out laser capture microdissection (LCM) based metabolomics on three A. lancea natural accessions with distinct AR colour patterns. Multivariate statistics across various SC types identified three polyacetylenes significantly associated with the red SCs of AR. Mass spectrometry imaging (MSI) further indicated that they are likely the causal compounds underlying the cinnabar-like colour of SCs in A. lancea. We thus provide a clear example of using spatial metabolomics to reveal key metabolite markers associated with important pharmaceutical properties by using very limited number of natural accessions in a non-model plant. It will further guide the elucidation of biosynthetic pathways for polyacetylenes in herbaceous plant A. lancea.