Ecological speciation is the process by which barriers to gene flow arise between populations as a result of ecologically based divergent selection. Environmental salinity has been identified as one of the most important ecological drivers influencing distribution, abundance, and species richness of aquatic organisms. Springs of the northern Chihuahuan Desert vary in salinity and are home to the Gammarus pecos (Crustacea:Amphipoda) species complex. We used field experiments to compare salinity tolerance among 9 amphipod populations from geographically discrete habitats differing in ambient salinities and to calculate salinity response distances among populations. Cluster analysis placed populations into 3 groups corresponding to low, medium, and high ambient salinities. Partial Mantel tests revealed significant positive correlations between salinity tolerance and habitat salinity after controlling for other variables, such as geographic distance and neutral genetic differences. Our results provide evidence that ecological speciation could be occurring among amphipod populations at different springs, as indicated by dissimilar physiological responses that are correlated with differences in ambient spring salinities. Gene flow is restricted among populations, the restriction is reinforced by dispersal barriers between springs, and selection might preserve variants that most effectively tolerate local salinity levels. Gammarus diversification in the northern Chihuahuan Desert is driven by vicariance and isolation, along with local selection and adaptation.