Johnsongrass is a troublesome weed infesting spring–summer crops. Poor control of johnsongrass after fluazifop-p-butyl treatments has been reported in central to northern Italy. Greenhouse and outdoor dose–response experiments revealed that four populations were highly resistant to fluazifop-p-butyl. All four were cross-resistant to other aryloxyphenoxypropionate (FOP) herbicides—propaquizafop, quizalofop, and haloxyfop. The resistance indexes ranged between 8 and 25 for propaquizafop and quizalofop, whereas a greater variability between populations was detected in response to haloxyfop. Conversely, cycloxydim and clethodim determined only a shift in the susceptibility with resistance index (RI) values of 2 to 3. Molecular analyses revealed that resistant plants possessed an insensitive acetyl coenzyme-A carboxylase (ACCase) target enzyme due to an Ile-to-Asn substitution at codon 2041. To our knowledge, this is the first report of such a mutation endowing ACCase resistance in johnsongrass. A molecular marker (CAPS assay) was developed for its rapid detection. Alternative mode of action herbicides S-metolachlor and nicosulfuron controlled all the FOP-resistant populations. Only a few chemical options are still available, and they have different efficacy on germinating seeds and sprouting rhizomes. To maintain efficacy over time, herbicides should be integrated with agronomic practices.