The northern acorn barnacle Semibalans banlanoides occupies several intertidal microhabitats which vary greatly in their degree of physical stress. This environmental heterogeneity creates distinct selection regimes which can maintain genetic variation in natural populations. Despite considerable attention placed on the link between spatial variation in fitness and balancing selection at specific loci, experimental manipulations and fitness estimates for molecular polymorphisms have rarely been conducted in the wild. The aim of this transplant experiment was to manipulate the level of physical stress experienced by a cohort of barnacles in the field and then investigate the spatial variation in fitness for genotypes at three loci: two candidate allozymes and the mitochondrial DNA control region. The viability of mannose-6-phosphate isomerase (Mpi) genotypes was dependent on the level of physical stress experienced in the various treatments; alternative homozygotes were favoured in alternative high stress-low stress environments. In contrast, the fitness of genotypes at other loci was equivalent among treatments and unaffected by the manipulation. Evaluated in the light of balancing selection models, these data indicate that the presence of multiple environmental niches is sufficient to promote a stable Mpi polymorphism in barnacle populations and that allelic variation at this locus reflects the process of adaptation to the heterogeneous intertidal landscape.