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The ubiquitous environmental fungus Aspergillus flavus is also a life-threatening avian pathogen.This study aimed to assess the genetic diversity and population structure of A. flavus isolated from turkey lung biopsy or environmental samples collected in a poultry farm.A. flavus isolates were identified using both morphological and ITS sequence features. Multilocus microsatellite genotyping was performed by using a panel of six microsatellite markers. Population genetic indices were computed using FSTAT and STRUCTURE. A minimum-spanning tree (MST) and UPGMA dendrogram were drawn using BioNumerics and NTSYS-PC, respectively.The 63 environmental (air, surfaces, eggshells and food) A. flavus isolates clustered in 36 genotypes (genotypic diversity = 0.57), and the 19 turkey lung biopsies isolates clustered in 17 genotypes (genotypic diversity = 0.89). The genetic structure of environmental and avian A. flavus populations were clearly differentiated, according to both F-statistics and Bayesian model-based analysis' results. The Bayesian approach indicated gene flow between both A. flavus populations. The MST illustrated the genetic structure of this A. flavus population split in nine clusters, including six singletons.Our results highlight the distinct genetic structure of environmental and avian A. flavus populations, indicative of a genome-based adaptation of isolates involved in avian aspergillosis.