Median lethal temperature (LT50) data from water-only exposures with the early life stages of freshwater mussels suggest that some species may be living near their upper thermal tolerances. However, evaluation of thermal sensitivity has never been conducted in sediment. Mussels live most of their lives burrowed in sediment, so understanding the effect of sediment on thermal sensitivity is a necessary step in evaluating the effectiveness of the water-only standard method, on which the regulatory framework for potential thermal criteria currently is based, as a test of thermal sensitivity. We developed a method for testing thermal sensitivity of juvenile mussels in sediment and used the method to assess thermal tolerance of 4 species across a range of temperatures common during summer. Stream beds may provide a thermal refuge in the wild, but we hypothesized that the presence of sediment alone does not alter thermal sensitivity. We also evaluated the effects of 2 temperature acclimation levels (22 and 27°C) and 2 water levels (watered and dewatered treatments). We then compared results from the sediment tests to those conducted using the water-only standard methods. We also conducted water-only LT tests with mussel larvae (glochidia) for comparison with the juvenile life stage. We found few consistent differences in thermal tolerance between sediment and water-only treatments, between acclimation temperatures, between waterlevel treatments, among species, or between juvenile and glochidial life stages (LT50 range = 33.3–37.2°C; mean = 35.6°C), supporting our hypothesis that the presence of sediment alone does not alter thermal sensitivity. The method we developed has potential for evaluating the role of other stressors (e.g., contaminants) in a more natural and complex environment.