Leonurus cardiaca (Motherwort) is one of the most resistant plants to biotic and abiotic stresses with a number of medicinal properties. In order to investigate the karyotypic variation between six populations of motherwort, this experiment was performed. Root-tip meristems were used for karyotypic evaluation. 8-hydroxy quinoline and ethanol–acetic acid (3:1 V/V) solution were used as pretreatment and fixative solutions, respectively. Chromosomes were stained by 2 % aceto-orcein. Long arm length (L), short arm length (S), total chromosome length (TL), L/S and S/L, overall genome length, the ratio of the longest to the shortest chromosome, and centromeric index were recorded. Result showed that the best root size for chromosome study of this species is 0.50–1.50 cm long and the best time for cutting the roots is about noon time. All the six studied populations were diploid (2n = 2x = 18), and metacentric chromosomes were dominant in all population. In some populations, a few submetacentric chromosomes were also observed. Cluster analysis based on karyotype traits revealed four groups of populations using Euclidean distance.
