We developed a set of molecular markers in Cistanche deserticola Y. C. MA to evaluate the production quality of cultivated C. deserticola individuals. This application utilizes the inter-simple-sequence repeat (ISSR) polymerase chain reaction (PCR) and random amplified polymorphic DNA (RAPD) PCR as molecular markers to determine the echinacoside content in cultivated C. deserticola individuals. The unweighted pair-group method using arithmetic average clustering (UPGMA) confirmed that the combined ISSR and RAPD data could categorize all C. deserticola individuals into three groups according to their respective echinacoside content. The stepwise multiple regression analysis (SMRA) revealed six potential markers associated with echinacoside accumulation in C. deserticola and produced 18 echinacoside-marker prediction models, four of which were successfully used to predict the quality of C. deserticola from Neimenggu. Both clustering and SMRA showed a correlation between the echinacoside content and molecular markers in cultivated C. deserticola. The relative average deviation of prediction (RADP) of the prediction models could be improved by simplifying and adjusting the model. It was found that the RADP value could reach 2.6% after adjustment and the simplified prediction models could successfully predict the quality of cultivated C. deserticola individuals.