Several inbred lines of acetolactate synthase (ALS)-inhibiting herbicide-resistant (ALS-R) Palmer amaranth and ALS-susceptible (ALS-S) common waterhemp were developed in the greenhouse. Interspecific hybrids were obtained by allowing several ALS-S common waterhemp females to be pollinated only by ALS-R Palmer amaranth in a growth chamber. Putative hybrid progeny were treated with an ALS-inhibiting herbicide, and the hybrid nature verified using a polymorphism found in the parental ALS gene. Polymerase chain reaction (PCR) was used to amplify a region of the ALS gene in both parental plants and putative hybrids. Restriction enzyme digestion of the ALS-R Palmer amaranth PCR fragment resulted in two smaller fragments, whereas the PCR fragment in the ALS-S common waterhemp was not cut. Restriction digestion of the putative hybrid PCR fragment showed a combination of ALS-R Palmer amaranth double fragments and an ALS-S common waterhemp single fragment. Approximately 4 million flowers were present on 11 common waterhemp females and produced about 44,000 seeds that appeared viable. From the approximately 3,500 putative hybrid seedlings that were screened, 35 were confirmed as hybrids using herbicide resistance as a phenotypic and molecular marker. The data collected here verify that interspecific hybridization does occur between these two species, and even at a low rate, it could contribute to the rapid spread of ALS resistance in these species.