Balancing selection describes evolutionary processes that maintain genetic diversity. To date, the number of impacted genes and underlying biological functions remain elusive. Using 60 three-spined stickleback genomes (Gasterosteus aculeatus) from five recently diverged lake-river population-pairs, we performed genome-wide scans across two levels of organization: population-pairs and populations. We overlapped Tajima’s D and Watterson’s estimator metrics and verified signals with additional summary statistics, and evaluated alternative explanations: neutral evolution, population structure, associated overdominance, or demographic change. Candidate windows exhibiting signals of balancing selection spanned 2.31% (population-pair) and 3.10% (population) of the autosomes. These candidate windows had extended linkage disequilibrium and were enriched in intergenic and non-synonymous SNPs. We identified 715 (population-pair) and 1,010 (population) candidate genes under balancing selection. Importantly, using conservative thresholds, we found a small proportion of candidate genes overlapped with highly differentiated loci or regions of potential associated overdominance. There was little evidence of confounding effects originating from demographic change. Overall, candidate genes under balancing selection were associated with functions related to interactions with the environment (olfaction and receptor signalling pathways). Our results demonstrate selection that maintains standing genetic variation is common and evolves in response to local environmental pressures, playing an important role in adaptation.