Methanogenic archaea are a genotypically and phenotypically diverse group that serve as key players in carbon cycling occurring in anaerobic environments. Comparative genomics of model methanogen strains have revealed a great deal of genetic variation among even the most closely related of sequenced methanogen genomes however, the ecological relevance of this variation in natural environments is largely unknown. The goal of this study was to investigate how genomic and ecological diversity was partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA), which vary in geochemical parameters and methanogen community composition.