Shattercane is a competitive weed in corn (Zea mays L.), soybean (Glycine max L.), grain sorghum and other crops in North America. Post-emergence control of shattercane in corn became possible with the introduction of acetolactate synthase -inhibiting (ALS) herbicides in the 1980’s, but their extensive use selected for populations of ALS-resistant shattercane. Shattercane seeds were collected in 1992 from 16 fields in southeastern and south-central Nebraska treated three consecutive years with primisulfuron. Four resistant plants were found in greenhouse evaluations of more than 30,000 plants. The objectives of this study were to isolate the ALS gene from shattercane and identify mutations in the ALS gene that confers herbicide resistance. Four ALS genes were amplified by PCR using primers derived from corn ALS sequence. The amplified genes were cloned, and sequenced from herbicide-resistant (mutant), sorghum, and -susceptible (wild type genotype). A total of eight nucleotides changes were detected in the four mutants compared to the susceptible shattercane wild type. A greenhouse bioassay was conducted to assess the response of each shattercane biotype to ALS-inhibiting herbicides from five chemical classes reported to have activity on shattercane or other grass weeds. There was a differential response among the biotypes to herbicides within and between chemical classes. One biotype was resistant or partially resistant to all the ALS-inhibiting herbicides applied. This biotype had a unique configuration of sequence changes relative to the other 3 biotypes, and will be crossed to adapted sorghum lines in an effort to develop additional sorghum lines that contain resistance to ALS-inhibiting herbicides.