Technical Abstract: Microbial populations within poultry litter have been largely ignored with the exception of potential human or livestock pathogens. A better understanding of the community structure and identity of the microbial populations within poultry litter could aid in the development of management practices that would reduce populations responsible for toxic air emissions and pathogen incidence. In this study, poultry litter air and physical properties were correlated to shifts in microbial community structure as analyzed by principal component analysis (PCA) and measured by denaturing gradient gel electrophoresis (DGGE). Litter samples were taken in a 36-point grid pattern at 5 m across and 12 m down a 146 m by 12.8 m chicken house. At each sample point, physical parameters such as litter moisture, pH, air and litter temperature, and relative humidity were recorded and samples were taken for molecular analysis. Poultry litter samples (0.3 g) were extracted in duplicate using Qbiogene’s Fast® DNA Spin kit for soil. DGGE analysis showed that the banding pattern of samples from the back and water/feeder areas of poultry house were distinct from those of samples from other areas. There were distinct clusters of banding patterns corresponding to the front, middle front, middle back, back, and water/feeder areas. PCA analysis showed similar cluster patterns, but with more distinct separation of the front, and mid-house samples. PCA analysis also showed that moisture content and litter temperature (accounting for 51.5% and 31.5% of the total variance, respectively) play a major role in spatial diversity of microbial community in the poultry house. Based on analysis of DGGE and 16S rDNA clones from DGGE band sequences, there appear to be differences in the types of microorganisms across the length of the house. These differences correspond to differences in the physical properties of the litter.