A giant ragweed population with putative resistance to cloransulam was identified in a long-term corn–soybean rotation located in southern Wisconsin. The population represented the first potential instance of giant ragweed resistance to acetolactate synthase (ALS) inhibitors in the state. Seeds were collected from several plants and pooled for subsequent experiments. Whole-plant dose–response experiments showed a high level of resistance (>500-fold) of the resistant (R) accession to cloransulam compared with a sensitive (S) accession. In vivo ALS bioassays showed that the target enzyme was 10.6- to 13.6-fold less sensitive to cloransulam in R than in S plants. Partial sequence analysis of the ALS gene found a tryptophan-to-leucine substitution at the 574 amino acid position (W574L) in the R phenotype. To better understand the potential fitness costs associated with the target-site substitution, replacement series experiments performed under greenhouse conditions characterized the relative growth, development, and fecundity of the R accession compared with an S accession in the absence of cloransulam. Growth over time did not differ between the R and S accessions for plant height during the vegetative phase (21 to 98 d after planting [DAP]) or for plant leaf area (21 to 80 DAP). At the estimated maximum, proportional shoot dry mass of each accession did not differ from theoretical proportions representing competitive equivalence, indicating no difference in vegetative competitive ability. Fecundity of R plants (430±53 seeds plant−1) did not differ from that of S plants (451±47 seeds plant−1), nor did seed viability (74 to 75% across accessions). This is the first report of equal competitive ability, fecundity, and seed viability between giant ragweed accessions R or S to cloransulam. The results suggest that the cloransulam resistance trait may persist and spread in the giant ragweed field population over time, even in the absence of selection by cloransulam.