Brooder’s pneumonia is a respiratory disease of poultry caused by fungi of genus Aspergillus. Aspergillus flavus is a toxigenic fungus, which sporulates easily in poor quality bedding and contaminates indoor air. The objective of present study is to isolate A. flavus from diseased birds, characterize toxigenic potential/variability and analysis of the risk factors associated with brooder’s pneumonia caused by A. flavus. Litter, air and morbid Cobb 500 strain from poultry farm (n=10) were selected for sampling. Moisture contents detected in bedding of saw dust and rice husk were 73 to 46.4 and 20.06 to 23.26% respectively. Poultry farms used saw dust were found more contaminated (CFU/30minutes 80±2.6 to 160±6.0) in comparison to those having rice husk (CFU/30minutes 28±3^a 50±2.6^b) as bedding material. A. flavus was isolated from lungs of all infected birds and identified based upon colony and microscopic morphology. Infected lung tissues containing fungal plaques were processed for histopathological examination. Tissue was found necrotized and contaminated with fungal hyphae. Purified A. flavus species were evaluated for aflatoxins (Total aflatoxins and AFB₁) production potential by Enzyme linked immunosorbent assay (ELISA). All species were found toxigenic with production of total aflatoxin ranged from 1.51 to 8.77ppm and AFB₁ from 0.33 to 3.0 ppm. Molecular analysis by Multiplex Polymerase chain reaction (PCR) revealed genetic variability in aflatoxins biosynthetic pathway. AflR gene was amplified in all species with 1032bps amplicon size. Multiplex PCR amplified the omt-A gene in 40, ver-1 in 70 and nor-1 in 60 percent toxigenic isolates. It is concluded that toxigenic A. flavus is emerging as major pathogen in Brooder’s pneumonia associated with poor litter quality.