AbstractSulfonylurea (SU) herbicides registered for use in other crops in Australia may leave residues in the soil interrupting rotations involving soybean. However, two unlinked genes (ALS1 and ALS2) confer SU tolerance in soybean. Here, we report experiments that explored the effectiveness of these ALS genes in Australian soybean germplasm. The first two experiments assessed the tolerance to halosulfuron‐methyl or metsulfuron‐methyl of lines with either zero ALS gene (ALS1) or both ALS1 and ALS2 in five different Australian genetic backgrounds. The presence of both ALS1 and ALS2 conferred strong SU tolerance at herbicide application rates as high as four times (4×) the recommended field rate, with evidence of interaction with the genetic background; lines carrying only ALS1 also performed well but demonstrated less tolerance. For the third experiment, the five lines homozygous for both ALS genes were intercrossed in a half‐diallel mating design. The resulting F2 population were screened for tolerance to metsulfuron‐methyl herbicide. In all instances, the presence of the two ALS genes provided absolute protection for the growth tip. Analyses of seedling dry weight and purple leaf discoloration demonstrated significant differences among the crosses. For dry weight, high and significant heritability estimates indicated significant additional genetic variation for SU tolerance in five of the 10 F2 crosses (p ≤ 0.05). The results of these three studies confirm that two ALS genes confer tolerance to SU herbicides when crossed into Australian soybean germplasm, but suggest possible epistatic effects within different backgrounds, and the possibility of other (minor) genes conferring additional SU tolerance.