Published records of twinning and superfoetation in monotocous wild mammals are rare. Flying-foxes (Pteropodidae, Megachiroptera) occasionally produce twin offspring, fraternal twins, as well as superfoetation twins. Superfoetation occurs where a foetus is conceived when there is a foetus already developing. The resultant twins may be months apart in developmental stages so that one twin is usually born prematurely. Here, we review the current literature available on twinning and superfoetation in flying-foxes, and describe nine occurrences of multiple conceptions in Australian Pteropus species. Differences in sex and age of offspring clearly showed that most resulted from simultaneous or serial ovulations, not zygote splitting, thus excluding monozygous twinning. Additionally, an example of superfoetation is genetically analysed using six highly polymorphic microsatellite loci, to show multiple-paternity of superfoetation twins. Multiple births by flying-foxes are rare, leading the authors to conclude that the polyovulation constraint theory, found in the Microchiroptera, is not applicable in flying-foxes. The rare occurrence of additional ovulations do not usually produce additional live offspring. Post-ovulatory constraints, including the extra energetic demand twins place on a female flying-fox, are implicated in preventing successful production of multiple offspring.