Bacteria excel in most ecological niches, including insect symbioses. A cluster of bacterial symbionts, established within a broad range of insects, share high 16S rRNA similarities with the secondary symbiont of the tsetse fly (Diptera: Glossinidae), Sodalis glossinidius
. Although 16S rRNA has proven informative towards characterization of this clade, the gene is insufficient for examining recent divergence due to selective constraints. Here, we assess the application of the internal transcribed spacer (ITS) regions, specifically the ITSglu
, used in conjunction with 16S rRNA to enhance the phylogenetic resolution of Sodalis
-allied bacteria. The 16S rRNA + ITS regions of Sodalis
and allied bacteria demonstrated significant divergence and were robust towards phylogenetic resolution. A monophyletic clade of Sodalis
isolates from tsetse species, distinct from other Enterobacteriaceae, was consistently observed suggesting diversification due to host adaptation. In contrast, the phylogenetic distribution of symbionts isolated from hippoboscid flies and various Hemiptera and Coleoptera were intertwined suggesting either horizontal transfer or a recent establishment from an environmental source. Lineage splitting of Sodalis
-allied bacteria into symbiotic and free-living sister groups was also observed. Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis
-allied symbionts in the field. These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis. These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.