Orobanche cumana Wallr. is a root parasite significantly affecting sunflower production in most cultivating countries. Due to the continuous introduction of resistant sunflower hybrids, the parasite evolves rapidly, developing new, more virulent races. In this context, extensive knowledge of the distinctive peculiarities of races and their pathogenicity is needed to understand the broomrape evolution and develop effective control strategies. The present study examines the comparative morphologic, genetic, transcriptomic, and proteomic analysis of two O. cumana accessions with different level of virulence (belonging to race E and H, respectively). Using simple sequence repeats (SSR) and inter-simple sequence repeats (ISSR) markers, specific amplicons suitable for distinguishing between races have been revealed. It has also been found that the genes PME, PGU, PRX, and CHS that encode enzymes associated with host invasion, such as pectin methylesterases, polygalacturonases, peroxidases, and chalcone synthases, respectively, were upregulated in race H compared to E at different developmental stages, confirming their role in the pathogenicity of broomrape. The proteomic analysis has showed 19 differentially expressed proteins (DEP), including 6 specific for race E and 13 specific for race H. A number of DEP were identified as enzymes of the respiratory system, cell wall-modifying enzymes, stress-related proteins, and other proteins known to be involved in the invasion of the host, and might be important for the more virulent race H. Obtained results provide new insight into broomrape parasitism and offer new opportunities for controlling this pathogen.