AbstractLake Sturgeon Acipenser fulvescens populations throughout the Great Lakes have experienced declines. Therefore, Lake Sturgeon have been stocked at several locations, including the Oswegatchie River in New York State, where Lake Sturgeon are listed as threatened. The goal of New York's Lake Sturgeon stocking program has been the establishment of self‐sustaining populations in six out of the seven management units in order to meet delisting criteria. Due to Lake Sturgeon's long lifespan and late sexual maturity, there is a significant delay before it can be determined whether natural reproduction is occurring at the reintroduction site. Our objective was to determine whether recently captured Lake Sturgeon from the Oswegatchie River were of hatchery origin and whether natural reproduction was occurring in the river. Age data and genetic data from 12 microsatellite loci were collected from 38 individuals from the Oswegatchie River. The genetic data were used to reconstruct families by using COLONY and STRUCTURE statistical analysis programs. The genetic diversity of the Oswegatchie River population was also compared with other natural and introduced Lake Sturgeon populations in the region. The combination of family reconstruction and age data indicated that natural reproduction is occurring in the Oswegatchie River. Individuals that were presumed to be recruited had ages ranging from 6 to 12 years. It is often thought female Lake Sturgeon typically do not reach sexual maturity until about 20 years of age. Only 12–15 years had elapsed since the earliest times of stocking, suggesting the possibility that remnant native females remained in the system or stocked female Lake Sturgeon have reached early sexual maturity. The genetic diversity of the Lake Sturgeon population in the Oswegatchie River is comparable to that of other nearby natural populations. However, the effective population size (Ne) was lower than that of many natural populations. As natural reproduction continues in the Oswegatchie River, Ne will likely increase.