Atractylodes lancea, A. chinensis, and A. macrocephala are the three most widely used medicinal species of the Atractylodes genus. Their similar morphological features cause disagreement as whether they are three unique species, leading to their frequent misuses in medical products. Our study aimed to understand their relationships through both the complete plastome sequences and nuclear sequences, to identify molecular markers for their differentiation and explore the evolutionary relationships among three species. We sequenced, annotated, and analyzed the plastomes of these three species. The plastomes are 153,201, 153,258, and 153,265 bps in length for A. lancea, A. chinensis, and A. macrocephaly, respectively. Similar to other Asteraceae species, their plastomes exhibit typical quadripartite structures. Each plastome consists of 119 distinct genes, including 78 protein-coding, 37 tRNA, and 4 rRNA genes. Analyses of indels, single-nucleotide polymorphisms and simple sequence repeats, and comparison of plastomes showed high degree of conservation, leading to difficulty in the discovery of differentiating markers. We identified eleven potential molecular markers using an algorithm based on interspecific and intraspecific nucleotide diversity gaps. Validation experiments with fifty-five individuals from the three species collected from the botanical garden and fields confirmed that the marker cz11 could effectively distinguish samples from the three different species. Analysis of the several nuclear sequences suggests that the species of A. macrocephala may be a hybrid of A. lancea and A. chinensis. In summary, the results from this study highlight the complex relationships among of these three medicinal plants.