Human-mediated animal movement can expose wildlife populations to novel environments. Phenotypic plasticity can buffer against the challenges presented by novel environments, while adaptation to local ecosystems may limit resilience in novel ecosystems. Outbreeding depression during the mixing of disparate gene pools can also reduce reproductive success after long-distance movement. Here, we use a ‘common-garden’ population of gopher tortoises (Gopherus polyphemus), translocated from numerous sites across the state of Florida, USA, to a mitigation site in the north-west (panhandle) region to assess whether geographic origin, outbreeding effects, and behavioral plasticity influence reproductive success in this threatened keystone species. We found that females from north-east Florida produced clutches with lower hatching success than females from other regions. We detected regional differentiation in nest site selection behavior in the common environment of the translocation site, though these differences did not mediate the regional effect on hatching success. We also found evidence for outbreeding depression: hatching success declined with increasing parental geographic and genetic distances, dropping from 93% to 67% across the range of observed parental genetic distances. Together, these results suggest that newly admixed populations may suffer reproductive costs due to historical population differentiation, and that undetected outbreeding depression could significantly hamper conservation efforts for this species and others undergoing a variety of human-mediated movements.