Although nest boxes have considerable application in the research and management of hollow-using arboreal marsupials, few studies have assessed whether species show a preference for particular nest box designs. This study aimed to determine whether the feathertail glider (Acrobates pygmaeus) showed a preference when offered four different designs, each of which had been used in earlier studies. We established one of each design in 45 plots across five locations, spanning north-east New South Wales and south-east Queensland. Feathertail gliders used 34 nest boxes at four locations but avoided a medium-sized rear-entry box with a 45-mm-diameter entrance. No clear preference was shown for three designs with narrow (≤25 mm) entrances. Feathertail gliders used 15 small rear-entry boxes, 10 large slit-entrance boxes and 9 wedge-shaped bat boxes. A slight preference was shown by breeding groups to occur in the non-wedge boxes compared with the wedge boxes. Squirrel gliders (Petaurus norfolcensis) used 51% of medium rear-entry boxes at four locations. This may account for the avoidance of this box design by feathertail gliders at these locations. Breeding and prolonged use of nest boxes by resident feathertail gliders and squirrel gliders at two locations in eucalypt plantations suggest that nest boxes can support local populations of arboreal marsupials where tree hollows are absent or scarce.