Negative anthropogenic influences on our freshwater ecosystems are increasing, as natural resources are exploited for our growing global population, resulting in increased contaminant loads to these fragile habitats. The expansions and proposals for new cross-continental oil and gas pipelines requires a need for a comprehensive understanding of how freshwater microbial populations function in hydrocarbon-rich environments. These baseline characterizations can provide insight into proposed bioremediation strategies crucial in cleaning up contaminated spill sites. Recent catastrophes and the increasing likelihood for pipeline fractures in the future suggest that this research is more pressing than ever. This study was conducted to reveal comparisons of in-situ microbial gene expression within freshwater hydrocarbon-rich reference sites cutting through the McMurray formation – the geologic strata constituting the oil sands. This is the first study to reveal metatranscriptomic comparisons in these freshwater ecosystems. Results confirm previously reported taxonomic variation, but now provide insight into the in-situ gene expression within these sites. Energy metabolism and hydrocarbon degradation genes are characterized, with emphasis on nitrogen, sulfur and methane processes, including transcripts relating to the observed expression of anaerobic methane oxidation. Expression of alkane monooxygenase (alkB) correlating to PAH concentrations at each site suggest it’s effectiveness as a bioindicator gene in freshwater environments. This information provides better linkages between natural and contaminated landscapes, closing knowledge gaps for optimizing not only oil sands mine reclamation but also understanding the biogeochemistry of other freshwater sites at risk of hydrocarbon contamination in the future.