Three suspected resistant (R1, R2, and R3) corn marigold populations collected from winter cereal fields located in central Greece were studied to confirm and elucidate the mechanisms of resistance to acetolactate synthase (ALS) inhibitors and their competitive ability against barley. Whole-plant dose–response assays proved that the three suspected R populations were highly cross-resistant to the ALS inhibitors tribenuron, pyroxsulam + florasulam, and imazamox, whereas their control with synthetic auxin plus ALS inhibitors co-formulated mixtures was increased in the order of tritosulfuron + dicamba < florasulam + clopyralid < tribenuron + mecoprop-P < florasulam + aminopyralid. The ALS gene sequence revealed a point mutation in 11 plants of the R1, R2, and R3 populations, which resulted in the substitution of Pro-197-Thr or Trp-574-Leu. By contrast, all three sequenced plants of the susceptible (S) population were found with the wild-type allele encoding Pro-197 and Trp-574. This is the first report of ALS-inhibitor resistance in corn marigold. The competition study between barley and four densities of the S, R2, or R3 populations indicated similar biomass rates for all three populations, suggesting lack of association between the competitive ability of the R populations and the target-site resistance mechanism, which was also confirmed by the similar biomass reduction rates of barley grown in competition with S or R populations.