Persicaria with 70–100 species in the world is distributed in temperate regions of both hemispheres. It has 11 species in Iran growing in moist areas and margins of rivers. Through hybridisation, plasticity and existence of overlapping habitats, species identification shows difficulty. In this study, we aimed to investigate karyotype characters and morphological features, evaluate genetic variability within and among species studied and examine species relationship using ISSR data. Nine annual taxa of Persicaria were gathered from different localities in Iran and used for studies. Our studies showed that combination of karyological, morphological and molecular data can delimit species studied. Based on karyological results, three chromosome counts (P. hydropiper (2n = 2x = 20), P. maculosa (2n = 2x = 22), P. orientalis (2n = 2x = 22)) were the first reports for the Flora of Iran. Analyses of morphological characters showed diagnostic features among taxa. STRUCTURE and AMOVA analyses showed high intraspecific genetic diversity. Our results suggested that phenotypic plasticity and hybridisation may cause genetic diversity within Persicaria species.