Genetic heterogeneity is a common trait in microbial populations, caused by de novo mutations and changes in variant frequencies over time. Microbes can thus differ genetically within the same species and acquire different phenotypes. For instance, performance and stability of anaerobic reactors are linked to the composition of the microbiome involved in the digestion process and to the environmental parameters imposing selective pressure on the metagenome, shaping its evolution. Changes at the strain level have the potential to determine variations in microbial functions, and their characterization could provide new insight into ecological and evolutionary processes driving anaerobic digestion. In this work, single nucleotide variant dynamics were studied in two time-course biogas upgrading experiments, testing alternative carbon sources and the response to exogenous hydrogen addition. A cumulative total of 76,229 and 64,289 high-confidence single nucleotide variants were discerned in the experiments related to carbon substrate availability and hydrogen addition, respectively. By combining complementary bioinformatic approaches, the study reconstructed the precise strain count-two for both hydrogenotrophic archaea-and tracked their abundance over time, while also characterizing tens of genes under strong selection. Results in the dominant archaea revealed the presence of nearly 100 variants within genes encoding enzymes involved in hydrogenotrophic methanogenesis. In the bacterial counterparts, 119 mutations were identified across 23 genes associated with the Wood-Ljungdahl pathway, suggesting a possible impact on the syntrophic acetate-oxidation process. Strain replacement events took place in both experiments, confirming the trends suggested by the variants trajectories and providing a comprehensive understanding of the biogas upgrading microbiome at the strain level. Overall, this resolution level allowed us to reveal fine-scale evolutionary mechanisms, functional dynamics, and strain-level metabolic variation that could contribute to the selection of key species actively involved in the carbon dioxide fixation process.