Understanding genetic evolution within species requires an accurate description of variation within and between populations and the ability to distinguish between the potential causes of an observed distribution of variation. In the cosmopolitan species Drosophila melanogaster, previous studies suggested that gene flow within and between continents is extensive and that most of the nuclear gene variation is found within, rather than among, populations. Here we present evidence that a population from Zimbabwe is more than twice as variable as those from the United States of America at the DNA sequence level, that most variants are not shared between the two geographic regions, and that there are nearly fixed differences between the Zimbabwe and USA samples in genomic regions experiencing low recombination rates. It appears that there is an unappreciated degree of population structure in D. melanogaster and that equilibrium models of molecular evolution are inappropriate for this species.