Chilean mussel populations have been thought to be panmictic with limited genetic structure. Genotyping-by-sequencing approaches have enabled investigation of genome-wide variation that may better distinguish populations that have evolved in different environments. We investigated neutral and adaptive genetic variation in Mytilus from six locations in southern Chile with 1,240 SNP obtained with RAD-seq. Differentiation among locations with 891 neutral SNPs was low (FST = 0.005). Higher differentiation was obtained with a panel of 58 putative outlier SNPs (FST = 0.114) indicating the potential for local adaptation. This panel identified clusters of genetically related individuals and demonstrated that much of the differentiation (~92%) could be attributed to the three major regions and environments: extreme conditions in Patagonia, inner bay influenced by aquaculture (Reloncaví́), and outer bay (Chiloé Island). Patagonia samples were most distinct, but additional analysis carried out excluding this collection also revealed adaptive divergence between inner and outer bay samples. The four locations within Reloncaví́ area were most similar with all panels of markers, likely due to similar environments, high gene flow by aquaculture practices and low geographic distance. However, fine scale structure could be detected when analyses included only this zone. Our results and the SNP markers developed will be a powerful tool supporting management and programs of this harvested species.