Fungicides in agriculture control disease and maintain crop productivity. However, resistant pathogen populations have emerged, weakening disease management strategies and threatening global food security. Using the SDHI fungicide, fluxapyroxad, and the causal agent of spot type net blotch in barley, Pyrenophora teres f. maculata (Ptm), as a fungicide-pathogen-crop system model, this study investigates the impact of fungicide distribution within plants in the selection pressure against fungicide resistant strains. Fluxapyroxad concentrations, applied as a seed coating, was mapped on different leaves at different growth stages using liquid chromatography coupled to high-resolution mass spectrometry. At the same time, the disease severity for fungicide susceptible and reduce sensitivity Ptm isolates was evaluated on individual leaves. Finally, a mixed-strain infection approach was employed to evaluate population shifts between susceptible and reduced-sensitivity Ptm isolates using digital PCR. Our findings enhance understanding of fungicide in planta behaviour and its influence on pathogen population, providing insights into the temporal progression of selection pressure and its implications for fungicide resistance development.