The maternally inherited bacterium Wolbachia infects the germline of most arthropod species. Using Drosophila simulans and D. melanogaster, we demonstrate that localization of Wolbachia to the fat bodies and adult brain is likely also a conserved feature of Wolbachia infection. Examination of three Wolbachia strains (WMel, WRiv, WPop) revealed that the bacteria preferentially concentrate in the central brain with low titres in the optic lobes. Distribution within regions of the central brain is largely determined by the Wolbachia strain, while the titre is influenced by both, the host species and the bacteria strain. In neurons of the central brain and ventral nerve cord, Wolbachia preferentially localizes to the neuronal cell bodies but not to axons. All examined Wolbachia strains are present intracellularly or in extracellular clusters, with the pathogenic WPop strain exhibiting the largest and most abundant clusters. We also discovered that 16 of 40 lines from the Drosophila Genetic Reference Panel are Wolbachia infected. Direct comparison of Wolbachia infected and cured lines from this panel reveals that differences in physiological traits (chill coma recovery, starvation, longevity) are partially due to host line influences. In addition, a tetracycline-induced increase in Drosophila longevity was detected many generations after treatment.