Whether and how habitat fragmentation and population size jointly affect adaptive genetic variation and adaptive population differentiation are largely unexplored. Owing to pronounced genetic drift, small, fragmented populations are thought to exhibit reduced adaptive genetic variation relative to large populations. Yet fragmentation is known to increase variability within and among habitats as population size decreases. Such variability might instead favour the maintenance of adaptive polymorphisms and/or generate more variability in adaptive differentiation at smaller population size. We investigated these alternative hypotheses by analysing coding-gene, singlenucleotide polymorphisms associated with different biological functions in fragmented brook trout populations of variable sizes. Putative adaptive differentiation was greater between small and large populations or among small populations than among large populations. These trends were stronger for genetic population size measures than demographic ones and were present despite pronounced drift in small populations. Our results suggest that fragmentation affects natural selection and that the changes elicited in the adaptive genetic composition and differentiation of fragmented populations vary with population size. By generating more variable evolutionary responses, the alteration of selective pressures during habitat fragmentation may affect future population persistence independently of, and perhaps long before, the effects of demographic and genetic stochasticity are manifest.