Genomic investigations of deep biosphere ecosystems have the potential for yielding ground-breaking insights into microbial evolution, but explicitly evolutionary studies are currently limited by technical constraints of environmental sequencing approaches and by the lack of a diverse collection of cultivated model organisms representing subsurface ecosystems (Biddle et al., 2012). This project aimed to study genome evolution in archaeal biofilms inhabiting carbonate chimneys of the Lost City hydrothermal field. The extremely high abundance and low diversity of these biofilms makes them attractive natural models for the study of genome evolution in deep biosphere habitats. The genomic characteristics of this unusual population structure have not yet been explored, so we began to optimize methods for the extraction and sorting of single cells from Lost City chimney biofilms in order to analyze single-cell genomic variability within and between biofilm communities. Our preliminary results indicated that enzymatic digestion of the biofilm extracellular matrix improves recovery of cells compared to typical cell extraction procedures that only utilize physical disruption of samples during cell extraction. We also developed a flow cytometric approach to evaluate the results of cell extraction procedures prior to investing resources into sorting and sequencing of single cells. Future data resulting from these efforts will allow us to pursue our broader goals of measuring the extent of genetic exchange between cells, viruses, and extracellular DNA in this rock-hosted, deep biosphere habitat.