Alum (Al₂(SO₄)₃•14H₂O) is a commonly used ammonia reducing poultry litter amendment that acidifies the litter to convert the volatile NH₃-N to the mineralized NH₄-N form. The effect of alum addition on the chemical makeup of the poultry litter has been previously studied, but very little work has been done to determine how alum addition affects the microbiology of poultry litter. In a laboratory scale study, replicate poultry litters were divided into 2 treatment groups (with or without alum addition), incubated at X °C for 16 weeks. To determine the effect of alum addition on the poultry litter microbiology, 16S (specific for bacteria) and 18S (specific for fungi) rDNA was amplified from the bulk community DNA extract, and community fingerprints were obtained using denaturing gradient gel electrophoresis (DGGE). To further determine the effect of alum addition on the bacterial communities, PCR primer sets specific for three bacterial groups known to be dominant in poultry litter (Actinomycetes, Clostridia, and Lactobacilli/Bacilli/Staphylococci) were used. Alum addition had significant effects on the bacterial and fungal communities, with the most drastic changes observed in the fungal communities. Alum addition resulted in the rapid increase in fungal diversity, which was prevalent throughout the remainder of the experiment, while the fungal population was practically absent in the non-alum treated litter by week 8. Shifts towards increased diversity were evident in the total bacterial community with alum addition, but not to the degree of the total fungal community. Analysis of the community structures of specific bacterial groups revealed small changes in the Actinomycetes populations, with little to no decrease in diversity as a result of the alum addition. Conversely, significant changes occurred in the both Low % G+C groups, with the Clostridia group showing a definite increase in diversity after the 12th week only in the alum treated litter.