The genetic diversity study of 39 broomrape populations from the Republic of Moldova was performed using 12 highly polymorphic SSR primer pairs, which shown the high level of polymorphism (average PIC value 0.57). We found that some of the SSR primers (Ocum-59 and Ocum-108) produced polymorphic bands suitable for discrimination between the studied populations. The diversity analysis within broomrape populations revealed a higher number of detected alleles and heterozygous loci in the accessions from the Southern region when compared to the Northern and Central ones. The average PIC values for the Northern, Central and Southern accessions ranged from 0.43, 0.48 to 0.56, respectively. Some populations from the Southern region (especially, Carabetovca, Alexanderfeld, Stefan-Voda and Slobozia Mare) have shown the major differences in the profiles obtained and presented the high level of genetic variability. The dendrogram based on genetic distance divided the 39 broomrape accessions into twelve clusters. High variability of O. cumana populations at molecular level was observed, which could be useful for further genetic studies and resistance-breeding programs.