Rhithropanopeus harrisii is a common estuarine crab native to the East and Gulf Coasts of North America. It is found along a broad range of salinities, ranging from ~1 PSU to ~25 PSU along estuarine gradients. As part of a larger study on the species' potential use of low-salinity refuges from parasitism, we tested for differences in gene expression with salinity using crabs from three distinct estuarine reaches along the Pamlico River in North Carolina. We acclimated crabs collected at natural sites with 1, 5 PSU, and 11 PSU mean salinity to 0.8, 3, and 15 PSU in the laboratory for one week and sequenced gill tissue (second posterior gill) from each group using mRNA-Seq.