•Ruscus hyrcanus is a rich source of medicinally important steroidal saponins.•Genetic structure of Ruscus hyrcanys populations was studied for the first time.•ISSR markers showed a significant difference among the twelve studied populations.•The most genetic diversity was intra-populational (Hs = 0.17).•The populations of RH6 and RH9 showed high pharmaceutical potential for further exploitation. Ruscus hyrcanus Woronow a commercially important ornamental and medicinal plant belonging to the Asparagaceae family, has various applications in Iranian traditional medicine due to its diuretic, appetizing, cathartic, vasoconstrictor, anti-hemorrhage, ant-nephritis, anti-infection, aperient and anti-varicose properties. In the present study, morphological, phytochemical and molecular markers were investigated in twelve R. hyrcanus (RH) populations collected from the Hyrcanian ecoregion in the north of Iran. Phytochemical characteristics were remarkably variable among the studied populations, but little morphological variation was observed. The highest contents of ruscogenin and neoruscogenin in the underground parts of the plant were recorded in populations RH9 (Abbasabad 3.41 mg/g DW) and RH6 (Darab Kola 1.44 mg/g DW), respectively. A total of 66 bands were obtained from 11 inter-simple sequence repeat (ISSR) markers, 62 (93.94 %) of which were polymorphic. Although the ISSR evaluation indicated an inter-population genetic differentiation (GST = 0.27), most of the genetic diversity was intra-populational (PPB = 32.26–75.81 %, He = 0.11−0.24, I = 0.17−0.36). A UPGMA cluster analysis of 120 samples of R. hyrcanus was carried out to categorize the studied populations, based on a molecular data matrix calculated with Jaccard’s similarity coefficient. A multiple regression analysis of the relationships between the ISSR bands and morphological and phytochemical traits demonstrated that eight ISSR fragments were related to ruscogenin in the plant materials. Several ISSR markers showed association with some morphological traits. This study provided the comprehensive preliminary data on morphological, phytochemical, and genetic characteristics of R. hyrcanus populations in order to exploit genetic reserves conservation programs.