Successful captive breeding programs are crucial to the long-term survival of many threatened species. However, pair incompatibility (breeding failure) limits sustainability of many captive populations. Understanding whether the drivers of this incompatibility are behavioral, genetic, or a combination of both, is crucial to improving breeding programs. We used 28 years of pairing data from the San Diego Zoo koala colony, plus genetic analyses using both major histocompatibility complex (MHC)-linked and non-MHC-linked microsatellite markers, to show that both genetic and non-genetic factors can influence mating success. Male age was reconfirmed to be a contributing factor to the likelihood of a koala pair copulating. This trend could also be related to a pair’s age difference, which was highly correlated with male age in our dataset. Familiarity was 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 MHC class II loci was associated with both pre- and post-copulatory female choice. Genome-wide similarity, and similarity at the MHC class II DAB locus, were also associated with female choice at the post-copulatory level. Finally, certain MHC-linked alleles were associated with either increased or decreased mating success. We predict that utilizing a variety of behavioral and MHC-dependent mate choice mechanisms improves female fitness through increased reproductive success. This study highlights the complexity of mate choice mechanisms in a species, and the importance of ascertaining mate choice mechanisms to improve the success of captive breeding programs.