Technical Abstract: Pyrosequencing can generate thousands of short sequences from DNA extracted from soil. These sequences can provide taxonomic information about the bacterial communities. We designed primers to the 16s rDNA and amplified DNA from soil samples from a long-term tillage/rotation trial in Kansas for two seasons. This 2 X 2 factorial trial had two rotation treatments (wheat-wheat and wheat-soybean), and two tillage treatments (conventional and no-till), with 4 replicate blocks sampled for each treatment. From the two years of sampling, a total of 20,180 DNA 16s rDNA sequences were generated and a total of 2337 operational taxonomic units (OTUs) were assembled using a 97% similarity cutoff. The most abundant taxonomic group was the phylum Acidobacteria. Other abundant groups were the phylum Gemmatimonadetes, the order Actinomycetales, and the class Alphaproteobacteria. Most of the Acidobacteria were identified to the group level with a high degree of confidence, and different groups showed a preference for different rotation/tillage treatments. For example, Group 1 showed a preference for continuous wheat versus wheat-soybean rotation, but group 4 showed a preference for wheat-soybean rotation. These results were validated with group specific primers and real-time PCR. This high-throughput sequencing approach can provide taxonomic information about the overall community, and detect community shifts resulting from cropping practices.