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 development of management practices that would reduce populations responsible for toxic air emissions. In this study, poultry litter physical, chemical and spatial properties were correlated to shifts in microbial community structure.  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, litter physical and chemical characteristics (temperature, moisture, pH, etc.) were determined and samples were taken for molecular analysis.  Poultry litter samples (0.3 g) were extracted in duplicate using Qbiogene’s Fast® DNA Soil extraction kit. This DNA extraction method gave the greatest yield of DNA (146 ± 66 ng ul^-1) and more distinct bands were obtained by denaturing gradient gel electrophoresis (DGGE) analysis of these samples.  Evaluation of total cell concentration by quantitative, real-time PCR analysis of the 16S rDNA showed that microbial cell concentrations were very high (3.0 ± 0.9 X 10¹⁰ cells gram^-1 litter). The total cell concentration doubled in the front of the house and in the feed and watering areas nearby where the chicks were concentrated and the litter was more moist.  DGGE analysis showed that the banding pattern of samples from this area was also distinct from those of samples from surrounding areas. Unique bands will be extracted and identified using 16S rDNA sequence analysis. Based on this data, there appear to be differences in the concentrations and types of microorganisms across the length of the house. These differences may be linked to litter physical properties and corresponding litter or house management regimes.