Genetic mixing aims to increase the genetic diversity of small or isolated populations, by mitigating genetic drift and inbreeding depression, either by maximally increasing genetic diversity, or minimising the prevalence of recessive, deleterious alleles. However, few studies investigate this beyond a single generation of mixing. Here, we model genetic mixing using captive, low‐diversity recipient population of the threatened Southern brown bandicoot (Isoodon obesulus) over 50 generations and compare wild populations across south‐eastern Australia as candidate source populations. We first assess genetic differentiation between 12 populations, including the first genomic assessment of three mainland Australian and three Tasmanian populations. We assess genetic diversity in the 12 populations using an individualised autosomal heterozygosity pipeline, using these results to identify a candidate recipient population for genetic mixing simulations. We found that populations fell into four major groups of genetic similarity: Adelaide Hills, western Victoria, eastern Victoria, and Tasmania, but populations within these groups were also distinct, and additional substructure was observed in some populations. Our autosomal heterozygosity pipeline indicated significant variability in mean heterozygosity between populations, identifying one extremely genetically degraded population on Inner Sister Island, Tasmania. Genetic mixing simulations of a low heterozygosity captive population in Victoria suggested the greatest increase in heterozygosity would be reached by using highly differentiated populations as mixing sources. However, when removing populations that may represent taxonomically discrete lineages, neither metrics of differentiation nor heterozygosity was strongly correlated with modelled heterozygosity increase, indicating the value of simulation‐based approaches when selecting source populations for population mixing.