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Successful captive breeding programs are crucial to the long-term survival of threatened species. However, pair incompatibility limits sustainability of many captive populations. Understanding whether the drivers of this incompatibility are behavioural or genetic, or a combination of both, is crucial to improving breeding programs. We used twenty-eight years of pairing data from the San Diego Zoo koala colony, plus genetic analyses using both MHC-linked and non-MHC-linked microsatellite markers, to show that both behavioural and genetic determinants can influence mating success. Male age was reconfirmed to be a contributing factor to the likelihood of a pair copulating. Familiarity was also reconfirmed to increase the probability of a successful copulation. Our data provided evidence that females select mates based on MHC and genome-wide similarity. Male heterozygosity at class II MHC loci influenced both pre- and post-copulatory female choice. Genome-wide similarity and similarity at the MHCII DAB locus were also found to influence female choice at the post-copulatory level. Finally, certain MHC-linked alleles were associated with increased or decreased mating success. We predict that utilising a variety of behavioural and MHC-dependent mate choice mechanisms improves female fitness through increased reproductive success. This study highlights the complexity of mate choice mechanisms within a species and the importance of ascertaining mate choice mechanisms to improve the success of captive breeding programs.