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 Wisconsin portions of the upper Mississippi River basin and compared the results to those from mussel populations experiencing a mortality event in the Clinch River in Virginia and Tennessee. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy bacterial species identified from the Clinch River was Yokenella regensburgei, which occurred in relatively high prevalence during the mortality event but was absent from samples acquired afterward. Its role in the mortality event, if any, is unknown but deserves further investigation. We suggest that future studies of freshwater mussel health incorporate hemolymph as a sample type due to its relative separation from the aquatic environment, its role in the circulatory system, and the fact that it can be collected nonlethally.