Background Sunflower broomrape ( Orobanche cumana Wallr.) is a holoparasitic plant that jeopardizes sunflower production in most areas of Europe and Asia. Recently, populations with increased virulence, classified as race G GV , have been identified in Southern Spain’s Guadalquivir Valley gene pool. These populations overcome resistance genes in hybrids resistant to the predominant race F GV . This study aimed to (i) determine the inheritance and map the avirulence trait segregating in a cross between O. cumana individuals from populations EK23 (F GV ) and IN201 (G GV ), and (ii) characterize the host effect on the IN201 parental population allelic diversity. Results A segregating population consisting of 144 F 2:3 families was evaluated for virulence using a differential sunflower genotype (Hybrid 1, resistant to race F GV and susceptible to race G GV ) and genotyped with SNP markers. The ratio of avirulent to virulent F 2:3 families was not significantly different to 1:3 (χ2 = 0.93; P = 0.34), indicating monogenic control of the avirulence/virulence trait. The Avr G−GV locus was mapped on the upper end of O. cumana chromosome 2, 9.2 cM distal from the SNP markers OS04791 and OS02805. Secretome analysis in the Avr G−GV region revealed a cysteine-rich CAP superfamily- and a glucan 1,3-beta-glucosidase family 3-encoding genes as possible candidates for Avr G−GV . SNP allelic analysis on the IN201 population parasitizing a highly susceptible genotype or the differential genotype Hybrid 1 showed that (i) IN201 structure was shaped towards virulent alleles at SNP loci linked to Avr G−GV (ii) there were significant allelic frequency differences associated with the host genotype at Avr G−GV –linked loci. Conclusions This study mapped for the first time an avirulence gene in parasitic plants using a classical genetic approach, confirmed a gene-for-gene model in the O.cumana –sunflower system, and showed the implication of this single avirulence gene in determining the structure of broomrape populations subjected to selection pressure posed by a resistant genotype. The results will contribute to a better understanding of the interaction between crops and weedy parasitic plants, and to effectively manage evolution of virulence by sustainable control strategies based on host genetic resistance.