Until recently, an enormous effort was needed to apply genomic tools to ecological investigations, especially when striving to uncover the functional mechanisms of phenotypic plasticity and the genetic basis of evolutionary adaptation within natural populations. This present study aimed to develop a genomic resource for an organism ideally suited for functional ecology and evolutionary research. Over 760 unique DNA fragments containing microsatellite loci were isolated and characterized from Daphnia to provide more than 500 molecular markers for constructing a genetic map and for localizing chromosomal regions containing genes of ecological importance via quantitative trait locus analyses. Although primarily developed to genotype members of the Daphnia pulex species complex, a significant fraction of these markers is potentially valuable for population genetics and recombination mapping of distantly related species. Over 60% of markers tested in cross-specific amplifications are possibly conserved within the subgenus Daphnia, whereas 48 and 18% of tested primers are found to amplify subgenus Hyalodaphnia and subgenus Ctenodaphnia DNA, which represents ∼140 and 200 million years of evolutionary preservation.