Common ragweed and common cocklebur plants were collected at two sites each in Illinois, Minnesota, and Ohio to analyze intraspecific variability of the gene encoding acetolactate synthase (ALS). A 385-nucleotide fragment within the coding sequence of ALS was compared among 24 plants of each of these two species from the six locations. Common ragweed ALS was highly variable, with polymorphisms observed at 48 (12.5%) of the 385 nucleotides among the 24 plants. Despite the numerous nucleotide polymorphisms, only two inferred amino acid polymorphisms were identified. No apparent population structure was suggested by the ALS sequence data, indicating widespread gene flow consistent with the wind-pollinated nature of common ragweed. In contrast to common ragweed, no ALS polymorphisms were identified among the common cocklebur plants used in this study. As a basis for comparing the extremes observed between common ragweed and common cocklebur, ALS intraspecific variability also was investigated in 10 plants each of tall waterhemp and smooth pigweed. Normalized to the number of plants analyzed, the number of nucleotide polymorphisms for both tall waterhemp and smooth pigweed was greater than that in common cocklebur but less than that observed in common ragweed. Information on variability of herbicide target-site genes may be useful in predicting the likelihood for herbicide-resistance development. However, all four of the species investigated in this study have evolved resistance to ALS-inhibiting herbicides, despite the different levels of ALS variability observed.