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PremiseParasitic plants and their hosts are model systems for studying genetic variation in species interactions across environments. The parasitic plant Striga hermonthica (witchweed) attacks a range of cereal crop hosts in Africa and exhibits substantial variation in performance on different host species. Some of this variation is due to local adaptation, but the genetic basis of specialization on certain hosts is unknown.MethodsTo identify genomic regions that are strongly differentiated between parasites attacking different host species, we present an alignment-free analysis of S. hermonthica population diversity using whole genome sequencing (WGS) data for 68 individuals from western Kenya. We validate our findings with germination experiments and analyses based on a de novo assembled draft genome.ResultsReference-free and reference-based analyses suggest that only a small portion of the S. hermonthica genome is strongly differentiated by host species in populations from western Kenya. Analysis of host-associated k-mers implicated genes involved in development of the parasite haustorium (a specialized structure used to establish vascular connections with host roots) and a potential role of chemocyanins in molecular host-parasitic plant interactions. Conversely, no phenotypic or genomic evidence was observed suggesting host-specific selection on parasite response to strigolactones, hormones exuded by host roots and required for parasite germination.ConclusionsThis study demonstrates the utility of WGS for plant species with large, complex genomes and no available reference. Contrasting with theory emphasizing the role of early recognition loci for host specificity, our findings support host-specific selection on later interaction stages, recurring each generation after homogenizing gene flow.