Restoring levels of genetic diversity in small and declining populations is increasingly being considered in biodiversity conservation. Evidence-based genetic management requires assessment of risks and benefits of crossing populations. Because risks are challenging to assess experimentally, e.g. through multi-generational crosses, decision-support approaches utilize proxy risk factors such as time since separation of lineages. However, the paucity of empirical datasets on fitness consequences of longer separation times tends to favour crossing lineages with conservatively short separations, restricting wildlife managers’ options. Here, we assessed the genetic outcomes of interbreeding in the wild between lineages of a threatened Australian freshwater fish (Macquarie perch) separated by an estimated 119,000–385,000 years of evolution in distinct environments. Fish belonging to the Murray-Darling Basin (MDB) lineage escaped from Cataract Dam—into which they were translocated in ~ 1915—into the Cataract River, where they interbred with the local Hawkesbury-Nepean Basin (HNB) lineage. Analyses of reduced-representation genomic data revealed no evidence of genetic incompatibilities during interbreeding of the two lineages in the Cataract River: assignment to genotypic clusters indicated a spectrum of hybrid types including second generation hybrids and backcrosses to both parental lineages. Thus, no adverse effects were detected from genetic mixing of populations separated by > 100,000 years. We are not advocating purposely crossing the two lineages for management purposes under present cost–benefit considerations, because there are currently sufficient intra-lineage source populations to beneficially mix. Instead, this study presents a useful calibration point: two morphologically different lineages evolved in different habitats for 119,000–385,000 years can successfully interbreed.
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