Background <p>Fungal pathogens are major contributors to global losses of crop yields. Despite large-scale efforts to develop fungicides and resistant plant genotypes, disease outbreaks still pose severe risks to food security due to fungicide resistance and high adaptability of fungal pathogens. Genetic mechanisms behind the acquisition of resistance and renewed virulence have been uncovered by genome sequencing, especially of pathogens of main crops targeted by major control programs. Here, we investigate the use of comparative genomics of historical isolates to investigate how the wider community of fungal plant pathogens evolved during agricultural intensification.</p> Results <p>We analysed historical cryopreserved fungal isolates from three species that were collected in the UK between 1950 and 2000. Comparative genomics of 32 genomes was used to identify variable genome regions that represent putative targets of strong selection during this period, focusing especially on targets of fungicides and putative effector genes that might underpin changes in virulence. Using methods suitable for isolate rather than population sampling, we found evidence of rapid changes in single nucleotide polymorphism frequency in a suite of genes involved in pathogenesis, which overlapped partly between two of the species. We also found turnover in effector gene content in the UK during the period, but generally conserved evolution of fungicide target genes. Sample time and host explained similar amounts of variation in both single nucleotide polymorphism (SNP) changes and variation in effector gene content.</p> Conclusions <p>The described approach could be scaled up in the future to reconstruct the evolution of hundreds of species and samples held in historical fungal collections worldwide throughout the course of the Green Revolution.</p>