The genus Amaranthus includes several important monoecious and dioecious weed species, and several populations of these species have developed resistance to herbicides. These species are closely related and two or more species often coexist in agricultural settings. Collectively, these attributes raise the concern that herbicide resistance might transfer from one weedy Amaranthus species to another. We performed research to determine if a dominant allele encoding a herbicide-insensitive form of acetolactate synthase (ALS) could be transferred from a monoecious species, A. hybridus, to a dioecious species, A. rudis. Numerous F(1) hybrids were obtained from controlled crosses in a greenhouse between A. rudis and herbicide-resistant A. hybridus, and most (85%) of these hybrids were herbicide-resistant. Molecular analysis of the ALS gene was used to verify that herbicide-resistant hybrids contained both an A. rudis and an A. hybridus ALS allele. Although hybrids had greatly reduced fertility, 42 BC(1) plants were obtained by backcrossing 33 hybrids with male A. rudis. Fertility was greatly restored in BC(1) progeny, and numerous BC(2) progeny were obtained from a second backcross to A. rudis. The herbicide-resistance allele from A. hybridus was transmitted to 50% of the BC(1) progeny. The resistance allele was subsequently transmitted to and conferred herbicide resistance in 39 of 110 plants analyzed from four BC(2) families. Parental species, hybrids, and BC(2) progeny were compared for 2C nuclear DNA contents. The mean hybrid 2C nuclear DNA content, 1.27 pg, was equal to the average between A. rudis and A. hybridus, which had 2C DNA contents of 1.42 and 1.12 pg, respectively. The mean 2C DNA content of BC(2) plants, 1.40 pg, was significantly (alpha < 0.01) less than that of the recurring A. rudis parent and indicated that BC(2) plants were not polyploid. This report demonstrates that herbicide resistance can be acquired by A. rudis through a hybridization event with A. hybridus.