Drosophila melanogaster and its close relatives have been extremely important model species in the development of population genetic models that serve to explain patterns of diversity in natural populations, a major goal of evolutionary biology. A detailed picture of the evolutionary history of these species is beginning to emerge, as the relative importance of forces including demographic changes and natural selection is established. A continuing aim is to characterise levels of genetic diversity in a large number of populations of these species, covering a wide geographic area. We have used collections from five previously un-sampled wild populations of D. melanogaster and two of D. simulans, across three continents. We estimated levels of genetic diversity within, and divergence between, these populations, and looked for evidence of genetic structure both between ancestral and derived populations, and amongst derived populations. We also investigated the prevalence of infection with the bacterial endosymbiont Wolbachia. We found that D. melanogaster populations from Sub-Saharan Africa are the most diverse, and that divergence is highest between these and non-Sub-Saharan populations. There is strong evidence for structuring of populations between Sub-Saharan Africa and the rest of the world, and some evidence for weak structure amongst derived populations. Populations from Sub-Saharan Africa also differ in the prevalence of Wolbachia infection, with very low levels of infection compared to populations from the rest of the world.