Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains. Author summary: The fungal pathogen A. fumigatus has a cosmopolitan distribution, which make its isolates genotypically and phenotypically diverse. The two commonly used reference clinical isolates, Af293 and CEA17, differ in growth, virulence and susceptibility to the immune system and drug treatment; however, their genomes are 99.8% identical with differences mainly at non-coding regions. The high genetic similarity between the two isolates implicates epigenetic controls for their physiological differences. Here, we analysed two chromatin modifications in the A. fumigatus genome and found that H3K4me3 profiles of the two isolates are similar, while H3K9me3 modification at the genome-wide level is highly variable. The H3K9me3 differences between Af293 and CEA17 are correlated with differences in transposon silencing and inactivation. In support of this, we observed independent gross chromosomal alterations in the Af293 strains of different laboratories, but not among CEA17 strains. Characterization of one of the Af293 variant strains, which has undergone chromosomal loss and amplification of two separate regions in chromosome VIII (Chr VIII), showed significant improvements in secondary metabolites production, growth and virulence compared to the original Af293 isolate. These results suggest that stochastic gross chromosomal changes occur more frequently in certain isolates. Overall, this study provides a link between epigenetic and genetic mechanisms for the adaptation of A. fumigatus to diverse environments. [ABSTRACT FROM AUTHOR]