Although transcriptomic analysis of wild organisms is a powerful tool to understand molecular differences among populations, most methods require the use of lethal sampling. In fish, the use of epidermal mucus is a promising method for development of nonlethal sampling tools. Previous studies have shown that mRNA is dynamically regulated in fish epidermal mucus following stressor exposure, suggesting that mucus is reflective of molecular changes occurring within the organism in response to its environment. The aim of the study was to determine whether transcriptomics of mucus could discern molecular differences among populations of lake trout. In order to do so, mucus was collected and sequenced from four geographically-distinct lake trout (Salvelinus namaycush) populations at the IISD Experimental Lakes Area. Principal component analysis (PCA) and hierarchical clustering of read data showed that each lake trout population had unique transcriptomic profiles, suggesting that RNA sequencing of mucus is able to discern molecular differences among fish populations. Furthermore, differential gene expression analysis identified regulation of immune-related transcripts and viral gene expression transcripts among populations. PCA and a mixed linear model of water quality parameters indicated that environmental variables accounted for transcriptomic variation among populations. However, 32% of transcriptomic variance was unaccounted for by the mixed linear model, suggesting that other variables may influence transcription, such as epigenetics and presence of pathogens. Overall, results indicate that RNA sequencing of epidermal mucus is an effective, nonlethal method to study transcriptional differences among fish populations and may be especially useful for studies of endangered species.