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In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ among different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (CP) and between chicken fecal DNA and an avian DNA composite consisting of turkey, goose, and seagull fecal DNA extracts (CB) to enrich for chicken-specific DNA fragments. A total of 471 non-redundant chicken metagenomic sequences were retrieved and analyzed. All of the clone sequences were similar to prokaryotic genes, of which more than 60% could not be assigned to previously characterized functional roles. In general terms, sequences assigned characterized functional roles were associated with cellular processes (11.7%), metabolism (11.0%) and information storage and processing (13.4%). Approximately 53% of the nonredundant sequences are similar to genes present in intestinal bacteria belonging to Clostridia (20.9%), Bacteroidetes (15.0%), and Bacilli (17.3%). Twenty-five sequences from the CP and CB clone libraries were selected to develop chicken fecal-specific PCR assays. These assays were challenged against fecal DNA extracted from 21 different animal species, including mammals and birds. The results from the host-specificity studies showed that 12 of the assays had a high degree of specificity to chicken feces. In addition, three assays were specific to chicken and turkey while another four assays tested positive to more than two avian species, suggesting a broader distribution of some of the enriched gene fragments among different avian fecal microbial communities. Fecal pollution signals were detected using chicken-specific assays in contaminated water samples, although the PCR assays showed different detection limits. These results indicate the need for multiple assays to detect poultry fecal sources of pollution. The competitive DNA hybridization approach used in this study can rapidly select for numerous chicken fecal metagenomic regions that can be used as potential genetic markers for fecal source tracking.