Lake sturgeon Acipenser fulvescens were extirpated from the St. Louis River Estuary (SLRE) by the early 1900’s due to overfishing and habitat degradation. A restoration stocking program began in 1983, and continued almost annually until 2000. Lake sturgeon stocked into the SLRE were primarily obtained from the Wolf River (Lake Winnebago) genetic stock (n = 861,000) but some sturgeon were obtained from the Sturgeon River (Lake Superior) genetic stock (n = 61,380). Recently, spawning and natural recruitment has been documented near the Fond du Lac Dam, the upstream limit for lake sturgeon migrating from Lake Superior. However, the genetic origin of lake sturgeon spawning in the SLRE was unknown. Our objectives were to determine (1) the genetic origins and (2) genetic diversity of lake sturgeon spawning in the SLRE. Using both GENECLASS2 and ONCOR, a majority (79–81%) of lake sturgeon captured in the SLRE during spawning (2016–2018) assigned to the Wolf River genetic stock (Lake Winnebago) with greater than 80% probability using established microsatellites and a standardized genetic baseline. Other genetic stocks present (≥1%) included the Pic and Goulais rivers and possibly the Black Sturgeon River (identified using GENECLASS2, but not ONCOR); no fish assigned to the Sturgeon River using either method. Genetic diversity metrics showed that the SLRE lake sturgeon population was similar to other Lake Superior lake sturgeon populations. Overall, the SLRE Sturgeon population appears headed towards recovery. Adaptive management practices currently being employed should be continued to help guide further recovery of this population.