Several Australian arboreal marsupials have been reported to show variation in mating system across populations, but most previous studies have not included genetic analyses to confirm the observations. Our aim was to test the hypothesis that monogamy was the predominant mating system in a population of yellow-bellied gliders (Petaurus australis) in south-western Victoria, using a combination of behavioural observations and molecular genetic analyses. Home-range overlap, cohesiveness of pairs, rates of den site co-occupancy and location of den trees within the home ranges of 13 adult gliders were determined via radio-tracking. A monogamous social system predominated, demonstrated by extensive home-range overlap between cohabiting adult males and females (40–100%) and little home-range overlap between adjacent territories (<14%). Males spent ~55% of their active time within 25 m of their female partners and 55–85% of their sleeping time in dens with their female partner. The parentage of all juveniles spotlighted within the 400-ha study area was analysed using five microsatellite DNA markers. Of 37 individuals genotyped, eight of 13 juveniles were assigned both social parents as true parents with ≥80% confidence. These results suggest that social and genetic monogamy predominated in this population of yellow-bellied gliders.