Little empirical work in nature has quantified how wild populations with varying effective population sizes and genetic diversity perform when exposed to a gradient of ecologically important environmental conditions. To achieve this, juvenile brook trout from 12 isolated populations or closed metapopulations that differ substantially in population size and genetic diversity were transplanted to previously fishless ponds spanning a wide gradient of ecologically important variables. We evaluated the effect of genome-wide variation, effective population size (Ne), pond habitat, and initial body size on two fitness correlates (survival and growth). Genetic variables had little effect on either fitness correlate, which were determined primarily by habitat (pond temperature, depth, and pH) and initial body size. These results suggest that some vertebrate populations with low genomic diversity, low Ne and long-term isolation can represent important sources of variation and be capable of maintaining fitness in, and ultimately persisting and adapting to, changing environments. Our results also reinforce the paramount importance of improving available habitat and slowing habitat degradation for species conservation.