The utility of DNA molecules in identifying and characterizing intestinal microorganisms depends on methods that facilitate access to DNA of sufficient purity, quantity, and integrity. An efficient and unbiased extraction of DNA is thus critical to the validity of the subsequent analysis of the prevalence and diversity of the DNA sources in the sample. The highly heterogeneous composition of the diet of vertebrates makes DNA isolation challenging for this environment. Here, we consider the key steps involved in DNA isolation from vertebrate gut microflora including sample homogenization, lysis of bacterial cells, and extraction and precipitation of DNA. A detailed protocol for DNA isolation of the microbial contents of intestine and feces is also provided. In addition, we refer to commercially available methods for DNA extraction from the vertebrate gut flora.