JERUSALEM artichoke is an important crop for a wide range of agricultural, medicinal and industrial purposes. Its tubers are rich in inulin and it is cultivated for food and animal feed. The current study aimed to develop an applicable protocol for micropropagation of three cultivars of Jerusalem artichoke (i.e., Balady, Fuza and Alba) using stem node explants. These explants were cultured on ½ MS medium augmented with different concentrations of kanamycin or cefotaxime. The lowest contamination correlated with highest survival percentages were recorded for ½ MS supplemented with 62.5mg L^-1 cefotaxime. For shoot multiplication, the maximum number of shoots (11) were obtained for Alba cultivar cultured on MS fortified with 1mg L^-1 BA + 0.1mg L^-1 NAA + 50mg L^-1 nano selenium. For rooting in vitro, the maximum values of rooting percent, root numbers/plantlet and length of roots were observed with ½ MS + 2mg L^-1 IBA + 0.1mg L^-1 NAA + 0.5mg L^-1 KIN. Resulted in vitro regenerated plantlets, were acclimatized on perlite and peat moss mixture (1:1), which gave the highest percent of survival (100%) for Alba followed by Fuza and Balady (80 and 60%, respectively). Moreover, the molecular characterization based on RAPD, ISSR and SCoT techniques was carried out. The polymorphic percentages among in vivo shoots of the three cultivars recorded 38.09, 42.3 and 34.61%, respectively; 61.53, 67.8 and 50% between in vivo shoots, in vitro regenerates and stem derived calli of the three cultivars. The dendrogram analysis of combined techniques showed high similarity between Balady and Fuza followed by Alba.