The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has long been recognized as a problematic environmental pollutant, and efforts to remediate contaminated soils, sediments, and groundwater have been going on for decades. In recent years, much interest has focused on using bioremediation to clean up these sites. The current study investigated the microorganisms (16S rRNA genes, Illumina) and functional genes (xenA, xenB, and xplA) linked to RDX biodegradation in microcosms composed of sediment or groundwater from two Navy sites. For this, experiments included sediment samples from three depths (5 to 30 ft) from two wells located in one Navy site. In addition, the groundwater upstream and downstream of an emulsified oil biobarrier was examined from another Navy site. Further, for the groundwater experiments, the effect of glucose addition was explored. For the sediment experiments, the most enriched phylotypes during RDX degradation varied over time, by depth and well locations. However, several trends were noted, including the enrichment of Pseudomonas, Rhodococcus, Arthrobacter, and Sporolactobacillus in the sediment microcosms. For the groundwater-based experiments, Pseudomonas, unclassified Rhodocyclaceae, Sphingomonas, and Rhodococcus were also highly abundant during RDX degradation. The abundance of both xplA and xenA significantly increased during RDX degradation compared to the control microcosms for many treatments (both groundwater and sediment microcosms). In a limited number of microcosms, the copy number of the xenB gene increased. Phylotype data were correlated with functional gene data to highlight potentially important biomarkers for RDX biodegradation at these two Navy sites.