Bacteroides molecular markers have been used to identify human fecal contamination in natural waters, but recent work in our laboratory confirmed cross-amplification of several human-specific Bacteroides spp. assays with fecal DNA from fish. For identification of unique molecular markers, Bacteroides from human (n = 4) and fish (n = 7) fecal samples were cultured and their identities were further confirmed using Rapid ID 32A API strips. The 16S rDNA from multiple isolates from each sample was PCR amplified, cloned, and sequenced to identify unique markers for development of more stringent human-specific assays. In human feces, Bacteroides vulgatus was the dominant species (75% of isolates), whereas in tilapia feces, Bacteroides eggerthii was dominant (66%). Bacteroides from grass carp, channel catfish, and blue catfish may include Bacteroides uniformis, Bacteroides ovatus, or Bacteroides stercoris. Phylogenic analyses of the 16S rRNA gene sequences showed distinct Bacteroides groupings from each fish species, while human sequences clustered with known B. vulgatus. None of the fish isolates showed significant similarity to Bacteroides sequences currently deposited in NCBI (National Center for Biotechnology Information). This study expands the current sequence database of cultured fish Bacteroides. Such data are essential for identification of unique molecular markers in human Bacteroides that can be utilized in differentiating fish and human fecal contamination in water samples.