A rapidly growing body of evidence has shown that non-coding small RNAs (sRNAs) regulate a variety of important biological processes across all domains of life, including bacteria. The sRNA Pxr in the model myxobacterial species Myxococcus xanthus functions as a developmental gatekeeper that prevents the initiation of fruiting body development until nutrients have been depleted. My dissertation research has focused on the origin and evolution of Pxr and its associated regulatory network in the myxobacteria. Using a combination of phylogenetic and molecular-genetic approaches, I tracked the origin of Pxr and examined its evolution at both sequence and functional levels in the myxobacteria. I showed that Pxr appears to have a single origin at the base of the suborder Cystobacterineae within the Myxococcocales (myxobacteria) order. Homologs of pxr are highly conserved and may play a common fundamental role in regulating fruiting body formation across diverse species of myxobacteria. Nevertheless, pxr duplications occurred in the genus Cystobacter and the specificity of its function may be evolving in these lineages. Further, following from a previous mutagenesis screen to identify genes involved in the Pxr regulatory pathway, I identified four genes that appeared to have important roles in the Pxr pathway. I characterized the evolutionary divergence of these genes across species and functional roles of some of these genes in Pxr synthesis, processing or function in M. xanthus. Finally, I discovered new pxr duplications in several Cystobacter species from whole-genome sequence data that had not been previously identified. Taken together, my research characterizes the evolutionary origin and diversification of a bacterial sRNA, a class of regulatory elements that has great importance in the function and evolution of bacterial genomes. This work also provides insights into the evolution of developmental gene regulation in prokaryotes.