Experiments were conducted to identify acetolactate synthase (ALS, EC 220.127.116.11 [formerly EC 18.104.22.168]) mutation sites in eight biotypes of smooth pigweed and correlate these mutations with patterns of herbicide cross-resistance. Four herbicide-resistant smooth pigweed biotypes (R5, R6, R7, R8) collected from fields in Virginia, Delaware, and Maryland, showed a similar response to postemergence applications of the ALS-inhibitors imazethapyr, pyrithiobac, chlorimuron, thifensulfuron, and cloransulam. These R biotypes ranged from 261- to 537-fold resistant to imazethapyr and 29- to 88-fold resistant to pyrithiobac. The biotypes also had reduced sensitivity to chlorimuron and thifensulfuron of 2- to 14-fold and 10- to 25-fold, respectively, relative to a susceptible smooth pigweed biotype (S). Biotypes R6, R7, and R8 had reduced sensitivity of 3- to 10-fold to cloransulam relative to the S biotype, whereas R5 had increased sensitivity. All of these biotypes were found to have a serine to asparagine substitution at amino acid position 653, as numbered relative to the protein sequence of Arabidopsis thaliana. This stands in contrast to four other imidazolinone (IMI)-resistant smooth pigweed biotypes (R1, R2, R3, R4) that were collected from fields in Somerset County, Maryland. These biotypes were found to have an alanine to threonine substitution at position 122 of the ALS enzyme and were previously characterized at the whole-plant level with high-level resistance to IMI herbicides, increased sensitivity to pyrimidinylthiobenzoate and triazolopyrimidine sulfonanilide herbicides, and low to no cross-resistance to sulfonylurea herbicides.