All species are living in variable environments. integrating the impact of changing environments into plant-pathogen studies becomes essential. This integration is key to expanding our understanding of the evolutionary dynamics governing plant-pathogen interactions. After subjecting Zymoseptoria tritici to 500 generations of experimental evolution in vitro under various temperature regimes, we assessed the evolved lineages’ virulence levels across six wheat cultivars. Additionally, we performed whole-genome sequencing on ten evolved lineages to identify accumulated mutations Our study revealed a reduction in virulence among several evolved lineages, with variability based on the host genotype. We observed trans-lineage segregating mutations in the genome, encompassing both synonymous and non-synonymous mutations within the secretome. Intriguingly, despite 500 generations of experimental evolution, no loss of dispensable chromosomes was detected These findings suggest that the abiotic environment can significantly influence the dynamic evolution of the plant pathogen Z. tritici .