The digestive tract of animals contains a very large numbers of microorganisms with a high diversity. Traditionally, characterization of these microbial communities has relied on the ability to clonally culture each microorganism. With significant improvements in nucleotide sequencing technologies to economically obtain billions of bases, the study of genetic material recovered directly from environmental samples is becoming increasingly affordable. The investigation of microorganisms as a community regardless of their ability to be cultured has become reality. Using the metagenomic approach for analysis of chicken intestinal homogenates, we were able to greatly enhance the understanding of communities of microorganism in healthy and Runting Stunting Syndrome-infected chickens. In particular, comparative analysis of metagenomes from infected and noninfected chickens resulted in the identification of microorganisms as pathogen candidates. In this chapter, we demonstrate step-by-step how tools for comparative metagenomic analysis can facilitate the resolution of complex, multifactor-involved diseases.