Water management measures in the 1970s in the Netherlands have produced a large number of “resident” populations of three-spined sticklebacks that are no longer able to migrate to the sea. This may be viewed as a replicated field experiment, allowing us to study how the resident populations are coping with human-induced barriers to migration. We have previously shown that residents are smaller, bolder, more exploratory, more active, and more aggressive and exhibited lower shoaling and lower migratory tendencies compared to their ancestral “migrant” counterparts. However, it is not clear if these differences in wild-caught residents and migrants reflect genetic differentiation, rather than different developmental conditions. To investigate this, we raised offspring of four crosses (migrant ♂ × migrant ♀, resident ♂ × resident ♀, migrant ♂ × resident ♀, resident ♂ × migrant ♀) under similar controlled conditions and tested for differences in morphology and behavior as adults. We found that lab-raised resident sticklebacks exhibited lower shoaling and migratory tendencies as compared to lab-raised migrants, retaining the differences in their wild-caught parents. This indicates genetic differentiation of these traits. For all other traits, the lab-raised sticklebacks of the various crosses did not differ significantly, suggesting that the earlier-found contrast between wild-caught fish reflects differences in their environment. Our study shows that barriers to migration can lead to rapid differentiation in behavioral tendencies over contemporary timescales (~ 50 generations) and that part of these differences reflects genetic differentiation.