A putative resistant johnsongrass (Sorghum halepense L.) population from a cotton field in northern Greece was studied to determine the levels of resistance to acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides and elucidate related mechanisms of resistance. The johnsongrass population showed cross‐resistance to the ACCase‐inhibiting herbicides fluazifop‐p‐butyl, quizalofop‐p‐ethyl, and propaquizafop, with poor levels of control even with four times of the recommended rates of these herbicides. However, the johnsongrass population was highly sensitive (100% control) to the ACCase inhibitors cycloxydim and clethodim and to the acetolactate synthase inhibitor nicosulfuron. Amplification and sequencing of the ACCase gene fragment from johnsongrass plants revealed an Ile to Asn substitution at codon 2041 of the ACCase enzyme. This is the first record of a molecularly confirmed ACCase‐resistant johnsongrass population from cotton fields in Europe. These findings highlight a threat against the sustainable use of ACCase‐inhibiting herbicides in controlling johnsongrass and other grasses in cotton fields.