The diagnosis of bacterial disease in freshwater unionid mussels has been hindered by a lack of baseline information regarding the microbial communities associated with these animals. In this study, we cultured and identified bacteria from the hemolymph of stable mussel populations from the upper Mississippi River basin and compared results to mussel populations associated with a mortality event in the Clinch River, VA and TN. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy isolate was identified from the Clinch River. Yokenella regensbergei was found with relatively high prevalence during the mortality event but was absent from post-mortality samples. Its role, if any, in the mortality event is unknown and deserves further investigation. We suggest that future studies of freshwater mussel health utilize hemolymph samples due to its relative disconnect with the aquatic environment, role in the circulatory system and nonlethal nature of collection.