We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60°C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl a. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new “red” cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat.