We report the first study of gut-associated bacteria of bark beetles using both culture-dependent and culture-independent methods. These insects are major pests of pine trees but also contribute to important ecological functions such as nutrient cycling. We found members of the α- and γ-Proteobacteria and Firmicutes in larvae of the southern pine beetle, Dendroctonus frontalis Zimmermann. Sequences from three larval guts were grouped into one to three operational taxonomic units (OTUs) at 3% difference among sequences. Communities in adult southern pine beetle guts consisted solely of members of the γ-Proteobacteria. These could be grouped into three to five OTUs at 3% difference between sequences. These gut communities have relatively low species richness, which may reflect the specialization needed to exploit a nutrient-poor food source, colonize a chemically complex habitat, and maintain consistent associations with mutualistic fungi. However, there is considerable variation in gut microbiota composition among individual insects, suggesting the need for additional studies on sources of variation and potential substitutability among species performing similar functions.