Trempealeau National Wildlife Refuge is a 6,226-acre (25.20 km2) National Wildlife Refuge located along the Upper Mississippi River in extreme southern Buffalo County and extreme southwestern Trempealeau County in Wisconsin, United States. It is in part a wetland consisting of backwaters away from the Mississippi and Trempealeau River, and is a significant element of the Mississippi Flyway. It is part of the Driftless Area, a portion of North America which remained free of ice during the last ice age, creating in part the deep gorge of the Mississippi, quite visible from this refuge.