Background/Question/Methods The non-pathogenic microbes associated with insects are known to have important consequences for host nutrition, immunity, resistance to parasitoids, and even resistance to human control efforts using biological insecticides. However, despite this ubiquitous relationship, few systematic surveys have been done within closely related yet ecologically diverse insects to explore their bacterial communities and link specific bacteria with host diet, genotype or geography. To study the interaction of host, diet and microbiome, and to inform future experimental work, generalist and specialist Drosophila species were collected from a variety of natural substrates. Drosophila is an ideal insect lineage for this survey because of the diversity of substrates used as food, the cosmopolitan nature of several species, and the occurrence of several host shifts to previously unused, toxic resources. Additionally, in lab reared Drosophila melanogaster, genetic differences promote pathological gut bacterial assemblages, reducing host survival. Thus, the Drosophila model will allow ecologically relevant field studies to be combined with genetic dissection of the pathways interacting with resident bacteria and with fitness studies in a controlled laboratory setting. To capture the range of natural variation of internal bacteria within Drosophila, wild flies were collected from natural substrates and immediately stored in ethanol for transport. After thorough external sterilization of fly bodies, total DNA was extracted with techniques known to optimize bacterial DNA concentration. Universal primers were used to PCR amplify the bacterial 16s rRNA gene. Ligation and cloning were performed and 48 to 96 clones from each sample were sequenced at the Joint Genome Institute (Walnut Creek, CA). Results/Conclusions . Over a dozen species of Drosophila were collected. Substrates were selected to maximize the breadth of resources that are utilized in nature. At least two species each of mushroom feeders, flower feeders, and fruit feeders were obtained. To determine how diet affects the microbiome, the same Drosophila species were collected from different natural food sources. Additionally, different species found using the same resource were simultaneously collected to give insight into how host genotype affects the microbiome composition, given that all flies had access to the same food and naturally occurring bacteria. This survey has found extensive variation in bacterial communities both between diet and between host species and, combined with concurrent laboratory experiments, shows the utility of Drosophila for studying the interaction between host, diet and microbiome.