Aerva javanica is one of Egypt’s most important traditional medicinal plants used as antidiarrheal and anthelmintic medicine and recently as an anticancer agent. In this study, variations among ten populations of Aerva javanica in different sites in the Eastern Desert of Egypt were analyzed based on morphological and ecological attributes and molecular variation expressed by Inter-Simple Sequence Repeat (ISSR) markers. Morphological diversity was higher for populations in the Wadi El-Markh and Bir Abbady regions than others. The polymorphism revealed by ten ISSR primers was 79.4% among populations. Distance trees created using the results obtained from soil variables, morphological characterizations, and molecular data showed that the highest similarity was 0.974 between Populations 8 and 9, while the lowest similarity was 0.715 between Population 1 and Population 3 regions. In conclusion, the obtained data are important to design a plan for sustainable conservation of Aerva javanica as an important medicinal plant having a wide interspecific genetic variability within various populations.