We present here the most detailed molecular characterization of an evolutionary series to date. Whole genome evolution of microbial strains to select for traits of interest via adaptive evolution has the potential to be a useful tool for biotechnological purposes. However, the population structure during microbial adaptation to an environment has largely been uncharacterized. We developed a method to better understand the population structure of yeast evolving under a nutrient-limited condition using fluorescently marked strains that are otherwise isogenic. This method allowed us to unequivocally demonstrate for the first time that clonal intereference and multiple mutations affect the population structure in a eukaryote. Our method allowed us to isolate the subpopulations containing the beneficial mutants and identify the exact mutations in five of the beneficial mutants throughout the course of a 440 generation evolution. Transcriptome profiles of these beneficial mutants correlated with the mutations observed.