The Polygonati Rhizoma have generated significant market attention for their medicinal and culinary applications. However, morphological similarities and ambiguous species boundaries complicate the identification of genera and species, thereby impeding product development and utilization within Polygonatum sensu lato. Despite the widespread application of the chloroplast genome for taxonomic boundary revisions for Polygonatum s.l., a critical gap persist regarding their genomic applicability and the lack of standardized pipelines for developing species-specific molecular markers capable of rapid discrimination among species. This study aims to assess the effectiveness of chloroplast genomes in clarifying the current taxonomic status of the genera and species of Polygonatum s.l., and develop a reliable process for rapid identification of designated species from other species. A total of 21 chloroplast genomes were sequenced and assembled, and subsequent analyses included phylogenetic inference, multiple molecular species delimitation methods, and an automated screening framework were employed for subsequent analysis. Comparative analyses revealed relatively conserved chloroplast genomes, with notable variation limited primarily to the length of IR and LSC regions. By integrating multiple delimitation methods, the chloroplast genome validated 82.46% of the current classifications of Polygonatum s.l., demonstrating strong support (90.63%) for species represented by multiple sequences, yet only moderate support (70%) for those with single-sequence representation. Additionally, this study established and validated a scalable molecular marker development framework, spanning from identification of species-specific SNPs/InDels to the design of high-resolution molecular markers, illustrated through case studies involving Heteropolygonatum and three medicinally significant Polygonatum species.