Walleye (Sander vitreus) are an important commercial and recreational fish species in North America. Lake Winnipeg, Manitoba, supports the second largest freshwater fishery in Canada, with walleye being a focal species. In 2017, wild-caught walleye from six different locations in the Lake Winnipeg and Lake Manitoba basins were collected and sampled for gill tissue. One of the goals of a larger project examining movement patterns in Lake Winnipeg is to develop molecular tools for examining the effects of environmental stressors on walleye in Manitoba. We used gill tissue samples from individuals from the different sampling locations to develop a reference transcriptome for future studies examining the effects of environmental stressors on this species.