Freshwater mussels (order Unionida) play a key role in freshwater systems as ecosystem engineers and indicators of aquatic ecosystem health. The fauna is globally imperilled due to a diversity of suspected factors; however, causes for many population declines and mortality events remain unconfirmed due partly to limited health assessment tools. Mussel-monitoring activities often rely on population-level measurements, such as abundance and age structure, which reflect delayed responses to environmental conditions. Measures of organismal health would enable preemptive detection of declining condition before population-level effects manifest. Metabolomic analysis can identify shifts in biochemical pathways in response to stressors and changing environmental conditions; however, interpretation of the results requires information on inherent variability of metabolite concentrations in mussel populations. We targeted metabolites in the haemolymph of two common mussels, Lampsilis cardium and Lampsilis siliquoidea, from three Indiana streams (USA) using ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectroscopy. The influence of species, stream and sex on metabolite variability was examined with distance-based redundancy analysis. Metabolite variability was most influenced by species, followed by site and sex. Inter- and intraspecies metabolite variability among sexes was less distinct than differences among locations. We further categorized metabolites by occurrence and variability in mussel populations. Metabolites with high occurrence (Categories 1 and 2) included those indicative of energy status (catabolism versus anabolism; arginine, proline, carnitine, nicotinic acid, pantothenic acid), oxidative stress (proline, glutamine, glutamate) and protein metabolism (thymidine, cytidine, inosine). Metabolites with lower occurrence (Category 3) are constituents of assorted metabolic pathways and can be important biomarkers with additional temporal sampling to characterize their variability. These data provide a reference for future temporal (before/after) monitoring and for studies of stressor–metabolite linkages in freshwater mussels.