Reintroduction programs can benefit from optimisation of source populations to maximise genetic diversity. Here, we report an approach to guide genetic supplementation of founder individuals to maximise genetic diversity in a reintroduction program for a nationally threatened Australian ground-dwelling marsupial, the southern brown bandicoot (eastern subspecies), Isoodon obesulus obesulus. Following local extinction ~ 100 years earlier, founding individuals were reintroduced to Booderee National Park in south-eastern Australia over three years from the nearest viable wild population, approximately 250 km to the south. To assess and manage risks associated with low genetic diversity, we measured genetic diversity of the reintroduced population relative to the diversity across the subspecies’ range. The genetic diversity of the initial founder population was comparable with other sampled populations; most likely because the source population had high genetic diversity relative to other locations. We simulated scenarios of supplementation of the founder population from alternative candidate sources. We identified populations in the Melbourne region of Victoria as those that would yield the greatest increase in genetic diversity in the Booderee population. The simulated increase in diversity resulting from supplementation was accurately predicted by the pairwise FST comparison between the candidate and recipient population, but not diversity of the supplementation source itself. Genetic diversity is an important consideration in reintroductions; our study enables managers to make informed decisions to maximise the long-term persistence and genetic diversity of reintroduced populations.