The genetic diversity of 17 Cuscuta campestris ecotypes collected from different regions of Iran was assessed using ISSR and protein markers. Ten ISSR primers generated a total of 361 bands, of which 347 bands were polymorphic. PIC (polymorphism information content), based on ISSR and protein data, averaged 0.66 and 0.4 per primer, respectively. Cluster analysis and PCA plots derived from Dice’s similarity coefficientof the two-marker systems were highly concordant. The analysis of molecular variance allowed us to partition variation into: 81% (variance among populations) and 19% (variance within populations) based on ISSR data; and 85% (variance among populations) and 15% (within populations) for protein data. This high variation among ecotypes could be due to the high self fertilization, limited gene flow or the low rate of pollen and seed migration among ecotypes. Knowledge of the genetic variability of the weed acquired through using different molecular tools can be helpful in developing management programs in order to effective control of the weed in crop fields.