The genetic variation of a pathogen population is a pivotal component of pathogen evolution, having important implications for emerging diseases, nosocomial infections, and laboratory subculturing practices. Furthermore, it is undoubtedly altered during infection of a host. We address this issue using an insect-fungal model system to examine the influence of serial host passage on the genetic variation of a pathogen population. Using amplified fragment length polymorphism, a strain of the opportunistic fungus, Aspergillus flavus, showing initially 98% genetic similarity, was assessed for changes in genetic diversity during repeated passage through Galleria mellonella larvae and compared to that of a parallel population serially subcultured on artificial media. In two independent trials, the genetic diversity of the population passed through the insect dropped significantly, while the genetic variation of the population subcultured on media increased or remained unchanged. However, there were no changes in virulence or the production of protease or aflatoxin, indicating an apparent lack of selection. We suggest that the insect acted as a genetic bottleneck, reducing the genetic diversity of the A. flavus population. The ability of a host to produce a genetic bottleneck in a pathogen population impacts our understanding of emerging diseases, nosocomial infections, and laboratory subculturing practices.