Herbicide resistance has been reported in several Amaranthus species throughout the U.S. Because evidence exists of interspecies hybridization in some species of this genus, this study was conducted to determine whether acetolactate synthase (ALS)-inhibiting herbicide resistance could be transferred from Amaranthus palmeri to Amaranthus rudis through interspecific crosses. Plants of each species were grown in a growth chamber, and controlled interspecies crosses were made between ALS-resistant and -susceptible plants. A total of 15 putative hybrid plants were produced from an estimated 10,000 cross-pollinated flowers. Analysis of restriction enzyme digests of the ALS gene in which a single base substitution confers resistance inferred that herbicide resistance had been transferred from a resistant male A. rudis to the hybrid plant. Offspring of hybrid plants, backcrossed to the susceptible parent, survived herbicide treatment, demonstrating that herbicide resistance was transferred between species. DNA analysis also was performed using the amplified fragment length polymorphism (AFLP) technique between parental and putative hybrid plants. Several unique bands were found only in the hybrid.