The ability to counter periods of low humidity is an important determinant of distribution range in Drosophila. Climate specialists with low physiological tolerance to desiccation stress are restricted to the tropics and may lack the ability to further increase resistance through evolution. Although the physiological adaptations to desiccation stress are well studied in Drosophila and other ectotherms, factors underlying evolutionary responses remain unknown because of a paucity of genetic data. We address this issue by mapping evolutionary shifts in D. melanogaster under selection for desiccation resistance. Genomic DNA from five independent replicate selected, and control lines were hybridized to high density Affymetrix Drosophila tiling arrays resulting in the detection of 691 single feature polymorphisms (SFPs) differing between the treatments. While randomly distributed throughout the genome, the SFPs formed specific clusters according to gene ontology. These included genes involved in ion transport and respiratory system development that provide candidates for evolutionary changes involving excretory and respiratory water balance. Changes to genes related to neuronal control of cell signaling, development, and gene regulation provide candidates to explore novel biological processes in stress resistance. Sequencing revealed the nucleotide shifts in a subset of the SFPs and highlighted larger regions of genomic diversity surrounding SFPs. The association between natural desiccation resistance and a 463-bp region of the 5' promoter region of the Dys gene undergoing allele frequency changes in response to selection in the experimental evolution lines was tested in an independent population from Coffs Harbour, Australia. The allele frequencies of 23 SNPs common to the two populations were inferred from the parents of the 10% most and 10% least resistant Coffs Harbour flies. The frequencies of the selected alleles were higher at all sites, with three sites significantly associated with the resistant Coffs Harbour flies. This study illustrates how rapid mapping can be used for discovering natural molecular variants associated with survival to low humidity and provides a wealth of candidate alleles to explore the genetic basis of physiological differences among resistant and susceptible Drosophila populations and species.