Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis, with whole-transcriptome profiling, using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively un-impacted river, were significantly increased in the gills, liver and kidney (63, 34 and 19 fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome, using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts, and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.