In this study, we examined the phylogenetic relationship of the lineages within the Astilbe chinensis species complex, which has long been taxonomically controversial. For this, population genetic analyses were performed using 20 species-specific EST-SSR makers developed for four taxa in the A. chinensis complex, including the Korean endemic A. koreana (220 individuals representing 11 populations). The four taxa in the species complex, which could not be discerned using nrITS sequences, were clearly distinguished in the EST-SSR data. STRUCTURE, UPGMA and PCA analyses also supported the genetic segregation of the four taxa. AMOVA results showed higher genetic differentiation between A. chinensis and A. koreana (Rst = 0.508) than within the species (A. chinensis-Rst = 0.2, A. koreana-Rst = 0.103), indicating significant genetic divergence has occurred between these species. Analysis of 20 EST-SSR Quantitative Trait Loci (QTL) showed polyploidy (tetraploidy) in A. koreana population. In addition, A. rubra samples collected from Yunnan (China) formed a distinct genetic cluster. In conclusion, the EST-SSR data proved to be useful for recognizing distinct lineages in the A. chinensis species complex. Morphological and molecular analyses with expanded samples may provide more convincing evidence to settle down the taxonomic controversy surrounding the species complex. In addition, a cytological study is required to verify the chromosome number and ploidy level of A. koreana.