Corroborative data collected from 16S rRNA clone libraries, intergenic transcribed spacer (ITS) region clone libraries, and 16S rRNA hypervariable region tag pyrosequencing demonstrate microdiversity within single-species archaeal biofilms of the Lost City Hydrothermal Field. Both 16S rRNA clone libraries and pyrosequencing of the V6 hypervariable region show that Lost City Methanosarcinales (LCMS) biofilms are dominated by a single sequence, but the pyrosequencing data set also reveals the presence of an additional 1654 rare sequences. Clone libraries constructed with DNA spanning the V6 hypervariable region and ITS show that multiple ITS sequences are associated with the same dominant V6 sequence. Furthermore, ITS variability differed among three chimney samples, and the sample with the highest ITS diversity also contained the highest V6 diversity as measured by clone libraries as well as tag pyrosequencing. These results indicate that the extensive microdiversity detected in V6 tag sequences is an underestimate of genetic diversity within the archaeal biofilms.