Summary: Aspergillus flavus is a saprophyte that infects a wide range of hosts as an opportunistic pathogen. Some isolates produce aflatoxins, toxic and carcinogen mycotoxins that pose health concerns for both animals and humans. Factors driving genetic diversity within the strains of A. flavus remain poorly understood. Mycelia and conidia of Aspergillus flavus are predominately multinucleate and presumably haploid. But careful characterization of ploidy or nuclear condition in this species has not been reported. The overall objective of our study was to determine if nuclear condition and ploidy contribute to diversity and ecology of A. flavus. Nuclear condition was observed by labeling nuclei in different strains with two nuclear fluorescent markers (yellow, EYFP or cyan, ECFP Fluorescent Protein). Fusion of nuclei labeled with different fluorescent markers resulted in mycelia and conidia of three types: expressing only EYFP; expressing only ECFP; or expressing both EYFP+ECFP. Conidia containing nuclei expressing EYFP+ECFP were separated by Fluorescence.. Activated Cell Sorting (FACS) to assess heterokaryosis and ploidy. A few conidia were determined to contain one nucleus expressing yellow and cyan fluorescent markers in the same nucleus. Our findings suggest that although conidial are predominantly homokaryotic, a small percentage (below 10%) of the population could be heterokaryotic or diploid. Since the diploids formed in our study were stable, we hypothesized that populations of the fungus may have haploid nuclei, diploid nuclei or both. To address this hypothesis, we obtained putative diploid strains isolated from field studies and assessed the stability of ploidy under stress. To further study ploidy and nuclear condition in A. flavus, we exposed the putative diploid of natural isolates to high temperature (40°C) and examined the resulting colony sectors. We found that when grown under high temperature, the natural isolates formed two different sectors. Conidia from sectors were analyzed for ploidy by flow cytometry and found to be diploid in one sector. Whereas conidia from the other sector appeared as haploids. Haploid and the diploid cultures showed diversity of growth, nutrient utilization and pathogenicity on an insect model Galleria mellonella. The putative diploids (field isolates) were more aggressive than the stress induced diploids. Further, evidence of dikaryons was obtained in another species, Aspergillus nomius. We found that conidia of A. nomius IC1516 to be multinucleate, but the majority had two nuclei (dikaryons). This strain showed similarity to A. flavus in carbon utilization and virulence. Our results provide insight into the nuclear conditions and ploidy of A. flavus and their effects on fungal ecology.