The genetic basis of host preference has been investigated in only a few species. It is relevant to important questions in evolutionary biology, including sympatric speciation, generalist versus specialist adaptation, and parasite-host co-evolution. Here we show that a major locus strongly influences host preference in Nasonia. Nasonia are parasitic wasps that utilize fly pupae; Nasonia vitripennis is a generalist that parasitizes a diverse set of hosts, whereas Nasonia giraulti specializes in Protocalliphora (bird blowflies). In laboratory choice experiments using Protocalliphora and Sarcophaga (flesh flies), N. vitripennis shows a preference for Sarcophaga, whereas N. giraulti shows a preference for Protocalliphora. Through a series of interspecies crosses, we have introgressed a major locus affecting host preference from N. giraulti into N. vitripennis. The N. giraulti allele is dominant and greatly increases preference for Protocalliphora pupae in the introgression line relative to the recessive N. vitripennis allele. Through the utilization of a Nasonia genotyping microarray, we have identified the introgressed region as 16 Mb of chromosome 4, although a more complete analysis is necessary to determine the exact genetic architecture of host preference in the genus. To our knowledge, this is the first introgression of the host preference of one parasitoid species into another, as well as one of the few cases of introgression of a behavioral gene between species.