The number of herbicide-resistant populations of black-grass (Alopecurus myosuroides Huds.) in winter cereal crops in Europe is increasing, leading to serious restrictions in winter cereal production. The aim of this study was to evaluate the reaction of five A. myosuroides populations: four suspected as being herbicide-resistant and one susceptible to mesosulfuron + iodosulfuron-based herbicide (acetolactate synthase (ALS) inhibitor). Mutation events in domains A and B of the als gene, and the relationship between resistance to mesosulfuron + iodosulfuron and plant fitness in the early vegetative stage were also examined. The fitness was assessed on the basis of tillering, average leaf number, dry mass accumulation, chlorophyll content and photosystem II efficiency. The whole-plant bioassay confirmed resistance to ALS inhibitors in four A. myosuroides populations, and the level of reduction of biomass accumulation after herbicide treatment corresponded with the number of als gene mutations conferring resistance in the population. Non-treated populations did not differ significantly in biomass accumulation, chlorophyll content or photosystem II efficiency. However, particular ALS-resistant populations revealed more intensive tillering than the susceptible population. In the examined A. myosuroides populations, resistance conferring mutations (Pro197Thr and Trp574Leu) of the als gene were identified. These mutations have not been found in Poland before. In two populations there were some survivors after mesosulfuron + iodosulfuron treatment, where no mutation in the als gene was identified. The survival rate in these populations after herbicide treatment suggests that these populations were composed of individuals with target-site resistance (TSR) and nontarget-site resistance (NTSR) to mesosulfuron + iodosulfuron.