Environmental gradients are expected to promote the diversification and coexistence of ecological specialists adapted to local conditions. Consistent with this view, genera of phototrophic microorganisms in alkaline geothermal systems generally appear to consist of anciently divergent populations which have specialized on different temperature habitats. At White Creek (Lower Geyser Basin, Yellowstone National Park), however, a novel, 16S rRNA-defined lineage of the filamentous anoxygenic phototroph Chloroflexus (OTU 10, phylum Chloroflexi) occupies a much wider thermal niche than other 16S rRNA-defined groups of phototrophic bacteria. This suggests that Chloroflexus OTU 10 is either an ecological generalist or, alternatively, a group of cryptic thermal specialists which have recently diverged. To distinguish between these alternatives, we first isolated laboratory strains of Chloroflexus OTU 10 from along the White Creek temperature gradient. These strains are identical for partial gene sequences encoding the 16S rRNA and malonyl coenzyme A (CoA) reductase. However, strains isolated from upstream and downstream samples could be distinguished based on sequence variation at pcs, which encodes the propionyl-CoA synthase of the 3-hydroxypropionate pathway of carbon fixation used by the genus Chloroflexus. We next demonstrated that strains have diverged in temperature range for growth. Specifically, we obtained evidence for a positive correlation between thermal niche breadth and temperature optimum, with strains isolated from lower temperatures exhibiting greater thermal specialization than the most thermotolerant strain. The study has implications for our understanding of both the process of niche diversification of microorganisms and how diversity is organized in these hot spring communities.