Characterization of the microbiota of chicken is of current interest. The goals of the current study were to apply anaerobic isolation methods to comprehensively isolate and identify bacteria from the gastrointestinal tract (GIT) of chickens and their environment. Bacterial communities within the drinking water were dominated by Escherichia, whereas communities in litter were more representative of the cecum. The crop and small intestine (jejunum and ileum) were dominated by Lactobacillus and Enterococcus spp., and the cecum was dominated by Proteus spp. The collection of bacteria isolated was dominated by Enterococcus spp., Escherichia/Shigella spp., Lactobacillus spp., and Proteus spp.; however, many rare-occurring taxa were observed. These included members of the Clostridales and Clostridium spp., which were commonly isolated from the ileum and cecum. Bacteria isolated by enrichment and direct plating differed. The selective De Man, Rogosa and Sharpe agar was commonly associated with the isolation of Lactobacillus spp. and yielded the lowest diversity of all methods utilized. Increased diversity and increased frequency of isolating Clostridium spp., were observed in enrichments of blood and mucus or plating on Columbia agar supplemented with 10% blood and gentamicin. Bacteria isolated from this study provide source material for genomic and functional studies in chicken hosts.