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Summary Due to the steadily increasing number of putative herbicide-resistant weed populations, the demand for rapid in-season tests is rising. In this study, we introduce a new quantitative herbicide-resistance test system based on chlorophyll fluorescence imaging analysis of photosynthesis-related parameters. Susceptible and herbicide-resistant populations of Alopecurus myosuroides (black-grass) were cultivated in multiwell tissue culture plates containing nutrient agar and different dosages of fenoxaprop-P-ethyl and mesosulfuron+iodosulfuron. The maximum quantum efficiency of the PSII was measured 3 h after transplanting (HAT) and then for seven days every 24 h. Data of maximum quantum efficiency of the PSII were compared with standard whole-plant pot tests and molecular tests for target-site mutations. It was possible to fit dose-response curves and calculate corresponding resistance factors for ED90 for all populations tested using the chlorophyll fluorescence imaging. It was possible to distinguish between resistant and susceptible populations. The results of the chlorophyll fluorescence imaging corresponded well with the standard whole-plant pot tests in the glasshouse. However, populations with proved target-site mutations did not differ from other herbicide-resistant populations in the maximum quantum efficiency values of the PSII. We conclude that the chlorophyll fluorescence imaging provides reliable data on herbicide resistance for both modes of action tested in a shorter time and using less space, compared with standard whole-plant pot tests in the glasshouse.