Maize is grown in most regions of Uganda. Aflatoxin contamination of maize occurs in Uganda but there is lack of information on the distribution of Aspergillus flavus in major maize production regions of the country. The objective of this study was to determine the incidence and severity of Aspergillus flavus in major maize growing regions of Uganda. Hierarchical sampling procedure was used to randomly collect maize samples from all different fields in 16 districts in two cropping seasons of 2013. Samples were assayed for A. flavus incidence and severity. Results revealed significant (P<0.001) variation among regions and districts within regions for A. flavus incidence and severity. The highest A. flavus incidence and severity were recorded for Pallisa (74.2% and 4.8, respectively), one of the leading maize producing districts in Uganda. Among regions, the highest A. flavus incidence and severity were registered in eastern region at 62.4% and 4.6 respectively. These results reveal the presence of A. flavus and provide information on its distribution in the key maize producing regions in Uganda. There is need for regular monitoring of aflatoxin levels in maize grain from the major maize production districts in Uganda in order to establish proper aflatoxin management guidelines.