In Poland, knotweed is represented by three species – Reynoutria japonica Houtt., Reynoutria sachalinensis (F.Schmidt) Nakai, and a hybrid between two former species - Reynoutria x bohemica J. Chrtek & A. Chrtková. Brought to Europe as ornamental plants, R. japonica and R. sachalinensis began to appear outside of cultivation areas due to their impressive ability to propagate vegetatively by rhizome. Currently, all three are classified as highly invasive alien species. Rhizomes of R. japonica and R. sachalinensis are rich sources of biologically active compounds and have been used in traditional Chinese and Japanese medicine. However, only R. japonica is accepted as an herbal drug source in the pharmacopoeias (Polygoni cuspidati rhizoma et radix). There is no data on the medical use of Bohemian knotweed, but this hybrid species is frequently misidentified as R. japonica. Traditionally used morphological markers were, however, often reported as insufficient in raw material identification. Qualitative HPLC/DAD/ESI-HR-QTOF-MS analyses were used to determine the chemical composition of the collected Reynoutria samples. The UHPLC-QTOF-MS analysis revealed a total of 117 detected compounds belonging to carbohydrates, stilbenes, flavan-3-ols, procyanidins, anthraquinones, organic acids, and naphthalenes. Out of these, the abundance of 6 compounds was estimated quantitatively. Based on LC-MS data for 117 compounds, the cluster analysis dendrogram categorised the 15 accessions into two distinctive clusters, revealing unequivocal separation of the R. sachalinensis from other taxa representatives. This study intended to explore genetic identity, diversity, and population structure using sequence-related amplified polymorphisms (SRAP), and start codon targeted (SCoT) markers correlated with morphological traits and metabolic profiles in 15 collections of three knotweed taxa. Both the SCoT and the SRAP markers were located in the medium (0.1 to 0.25) or high (0.30 to 0.40) levels of PIC (polymorphic information content) and were considered as differentiating. Based on their polymorphic SRAP and SCoT fragments, the cluster analysis dendrogram categorized the 15 accessions into three distinctive clusters, closely referring to three knotweed taxa. The highest correlation between dendrograms was observed between genetic and morphological data, with values of 0.85 according to Baker’s methodology. Lower correlations were noted between genetics and metabolomics, and between morphology and metabolomics. The study suggests the effectiveness of morphological, phytochemical and molecular combinations employing SCoT and SRAP marker systems to assess the origin of plant material, and proves them useful for clarifying the population structure and assessing spread, diversity and evolution of the invasive species such as Reynoutria spp.