Local adaptation to novel environments is an important driver of divergence among closely related species. We have examined the process of local adaptation to distinct visual environments among the cisco species flock (Coregonus spp.) of Lake Superior. We developed a new protocol for long-read amplicon sequencing using the Oxford Nanopore Flongle device, and employed this pipeline to genotype five visual opsin genes from individuals of C. artedi, C. hoyi, C. kiyi, and C. zenithicus from Lake Superior. Results revealed high levels of differentiation in a key amino acid residue involved in the spectral tuning of rhodopsin (Tyr261Phe), with the allele for 261Phe fixed in C. kiyi. This species is typically found at depths of 80 to >200m, a blue-shifted light environment as compared to the surface. The concordance between C. kiyi’s preferred habitat and the predicted 8 nm blue-shift in rhodopsin’s absorption spectrum associated with the 261Phe allele provides compelling evidence that this species is adapted to life in deep water. Additionally, an ancestral reconstruction of the amino acid state at rhodopsin residue 261 across the fish tree of life has shown that the changes observed in Coregonus spp. independently parallel those found in other distantly related lineages. In certain lineages, it even appears that “toggling” back and forth between the two allelic states at this site has occurred over deep evolutionary time.