The lone star tick, Amblyomma americanum (Acari: Ixodidae), is commonly reported from people and animals throughout the eastern U.S. and is associated with transmission of a number of emerging diseases. To better define the microbial communities within lone star ticks, 16S rRNA gene based analysis using bacteria-wide primers, followed by sequencing of individual clones (n = 449) was used to identify the most common bacterial operational taxonomic units (OTUs) present within colony-reared and wild A. americanum. The colony-reared ticks contained primarily sequence affiliated with members of the genus Coxiella (89%; 81/91), common endosymbionts of ticks, and Brevibacterium (11%; 10/ 91). Similarly, analysis of clones from unfed wild lone star ticks revealed that 96.7% (89/92) of all the OTUs identified were affiliated with Coxiella-like endosymbionts, as compared with only 5.1–11.7% (5/98–9/77) of those identified from wild lone star ticks after feeding. In contrast, the proportion of OTUs identified as Rickettsia sp. in wild-caught ticks increased from 2.2% (2/92) before feeding to as high as 46.8% (36/77) after feeding, and all Rickettsia spp. sequences recovered were most similar to those described from the spotted fever group Rickettsia, specifically R. amblyommii and R. massiliae. Additional characterization of the Rickettsiales tick community by polymerase chain reaction, cloning, and sequencing of 17 kDa and gltA genes confirmed these initial findings and suggested that novel Rickettsia spp. are likely present in these ticks. These data provide insight into the overall, as well as the rickettsial community of wild lone star ticks and may ultimately aid in identification of novel pathogens transmitted by A. americanum.