The ability of hatchling and juvenile turtles to thermoregulate, as evidenced by selection of preferred temperatures in thermal gradients, is an important factor influencing fitness. Previous studies in laboratory thermal gradients suggest that most young turtles select warm temperatures (25°–33°C) within a narrow range. Confounding factors affecting microhabitat selection, such as predator avoidance, foraging, or social interaction, may influence thermal preference in some species, particularly softshell turtles (Apalone spp.) that frequently bury in the substrate to remain cryptic. We analyzed temperature selection of juvenile Apalone spinifera in an aquatic thermal gradient of 15°–30°C with either gravel, sand, or a combination of sand and no substrate. Across all substrate treatments in the gradient tests, hatchlings selected the warmest temperature available (30°C: 56.1% of observations) and avoided the 2 coldest temperatures (15°C: 5.3%; 18°C: 3.6%). Turtles relocated between chambers more often in control tests than when a gradient was present and fewer observations involved a chamber relocation in control tests. In gradient tests, chamber temperature significantly influenced selection, and the interactive effect of temperature and substrate was significant. We observed turtles more frequently in 30°C in tests conducted with a sand substrate than with a gravel substrate or without a substrate. In tests conducted with sand in the 4 coolest chambers only, turtles chose the warmest temperature with a sand substrate more often than all other temperatures, except 30°C. Our results indicate that hatchling A. spinifera can effectively detect temperature differences and select preferred temperatures within at least a 3°C range and that substrate type may affect selected temperature.