Symbiotic rhizobia in legumes account for a large portion of nitrogen fixation in the biosphere. Nitrogen fixation is an energy-demanding process requiring tight control of metabolism and redox state. It is of great interest to understand the bacteroid differentiation process and the roles of energy storage molecules, such as glycogen and polyhydroxybutyrate (PHB), in maintaining the Rhizobium-legume symbioses. Traditional biochemical assays for checking phenotypic changes of mutants require a large volume of starting materials, which is difficult for unculturable, terminally differentiated bacteroids. Here we present a label-free technique that allows the identification and characterization of phenotypic changes of bacteria at the single-cell level. This work demonstrates the application of single-cell Raman spectra (SCRS) to differentiate Rhizobium leguminosarum bv. viciae wild-type and mutants under different conditions. We found symbiotically differentiated bacteroids and free-living bacteria differed primarily at a Raman biomarker, cytochrome c, corresponding to a bacteroid-specific terminal oxidase. We demonstrated that, for the first time, SCRS were able to link phenotypic changes and specific genetic mutants, in this case, single and double mutations in synthesis of carbon storage molecules glycogen and polyhydroxybutyrate (PHB). By analyzing SCRS of these mutants, it provides insights into metabolite production and carbon regulatory network of rhizobia.