Water availability is a major environmental challenge to a variety of terrestrial organisms. In insects, desiccation tolerance varies predictably over various spatial and temporal scales and is an important physiological basis of fitness variation among natural populations. Here, we examine the dynamics of desiccation tolerance in North American populations of Drosophila melanogaster using: 1) natural populations sampled across latitudes and seasons in the eastern USA; 2) experimental evolution in the field in response to changing seasonal environments; 3) a sequenced panel of inbred lines (DGRP) to perform genome wide associations and examine whether SNPs/genes associated with variation in desiccation tolerance exhibit patterns of clinal and/or seasonal enrichment in pooled sequencing of populations. In natural populations we observed a shallow cline in desiccation tolerance, for which tolerance exhibited a positive association with latitude; the steepness of this cline increased with decreasing culture temperature, demonstrating a significant degree of thermal plasticity. No differences in desiccation tolerance were observed between spring and autumn collections from three mid-to-northern latitude populations, or as a function of experimental evolution to seasonality. Similarly, water loss rates did not vary significantly among latitudinal, seasonal or experimental evolution populations. However, changes in metabolic rates during prolonged exposure to dry conditions indicate increased tolerance in higher latitude populations. Genome wide association studies identified thirty-six SNPs in twenty-eight genes associated with sex-averaged drought tolerance. Among North American populations, genes associated with drought tolerance do not show increased signatures of spatially varying selection relative to the rest of the genome, whereas among Australian populations they do.