Standard laboratory sediment toxicity methods have been adapted for conducting toxicity tests with juvenile freshwater mussels. However, studies looking at juvenile mussel burrowing behavior at the water-sediment interface are limited. Juvenile mussels burrow in sediment for the first 0 to 4 yr of life but also may inhabit the sediment-water interface. The objective of this study was to evaluate burrowing behavior of various species and ages of juvenile freshwater mussels in three control sediments: West Bearskin Lake, Spring River, and coarse commercial sand. Species tested included (1) Fatmucket (Lampsilis siliquoidea), (2) Notched Rainbow (Villosa constricta), (3) Washboard (Megalonaias nervosa), (4) Rainbow (Villosa iris), (5) Arkansas Fatmucket (Lampsilis powellii), and (6) Oregon Floater (Anodonta oregonensis). Greater than 95% of the mussels burrowed into test sediment within 15 min. Across species, life stage, and substrate type, most mussels were recovered from the upper layers of sediment (91% at a sediment depth of 3.4 mm or less), and only 2% of the mussels were recovered at a depth >5.1 mm. No mussels were recovered from a depth >6.8 mm. There was no difference in mussel burrowing depth at 4 h versus 24 h across species, age, and sediment type. Two ages of Fatmucket burrowed to a significantly greater depth in the West Bearskin Lake sediment compared to the Spring River sediment or Coarse Sand. However, there was no significant difference in mean depth across sediment type with the other five species of mussels tested. Based on species and age of mussels tested, juvenile mussels up to an age of at least 20 wk and a length of at least 5 mm readily burrow into sediment and likely would be exposed to contaminants in whole sediment and associated pore water throughout a laboratory sediment toxicity test.