Lake sturgeon (Acipenser fulvescens) is a species of conservation concern that has been stocked in several Great Lakes (North America) rivers. Lake sturgeon were extirpated in the Ontonagon River in Lake Superior and stocking began in 1998. In 2017, gametes were collected from spawning lake sturgeon (9 females, 36 males) caught at the nearby Sturgeon River spawning ground, generating nine family groups using a 1:4 mating design (n = 862). In 2018, gametes were collected from 3 females and 15 males, generating three family groups, and additional collections of drifting fry from the Sturgeon River were reared in the hatchery, resulting in 84 hatchery-produced and 675 wild-caught fry for stocking in the Ontonagon River. The objective of this study was to compare paternal representation and genetic diversity between the two stocking strategies. Parentage analysis based on genetic data from 12 microsatellite loci determined none of the family groups in the hatchery had equal paternal representation (p < 0.001), while wild-produced offspring had equal paternal representation. Despite the larger number of breeders contributing to the wild-caught larvae, there was no significant difference in genetic diversity between the wild-caught larvae and representative hatchery-produced offspring.