The hoatzin is the only known avian species with foregut fermentation. It is a primarily folivorous feeder and has a distended crop and lower/distal esophagus, which has evolved for the microbial fermentation of ingested feed. Crop samples collected from 10 individual animals from the Apure River area, Apure State, Venezuela were examined for the presence and density of methanogens using 16S rRNA gene clone libraries and real-time PCR prepared from pooled and individual PCR products. A total of 197 clones were examined, revealing 24 different methanogen 16S rRNA sequences, or phylotypes. Of the 24 unique phylotypes, 16 (171 of 197 clones) formed five unique clades within the genus Methanobrevibacter with the largest group of clones (118 clones) 98.7% similar to Methanobrevibacter ruminantium. The remaining eight phylotypes (26 clones) formed four unique clades that had only 94.0-96.7% identity to Methanosphaera stadtmanae. Based upon 98% sequence identity, we identified 17 of the 24 methanogen phylotypes from the hoatzin as possible new species and strains, with three phylotypes representing possible new genera (<94.5% sequence identity). Although none of the hoatzin methanogen phylotypes had 100% sequence identity to any other archaeal sequences in the GenBank database, the hoatzin crop methanogen sequences formed sister groups with known rumen methanogens. Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using real-time PCR, were 5.80 x 10(9), 7.93 x 10(12) and 3.31 x 10(5), respectively. The crop microbial data presented here provide an excellent example of convergent evolution of foregut fermentation in the hoatzin, similar to that of ruminants.