Herpesvirus infections associated with a range of clinical findings are widespread in free-ranging and captive Australian marsupials. We report on herpesviruses identified by virus neutralization and PCR in free-ranging and captive Lumholtz's tree-kangaroos (Dendrolagus lumholtzi). Herpesvirus has not been confirmed previously by DNA testing in tree kangaroos. Virus neutralization testing for alphaherpesviruses MaHV1 and MaHV2 was positive on 4/10 captive and 0/35 free-ranging tree-kangaroo samples tested. A novel gammaherpesvirus was found on PCR in 17/20 apparently healthy individuals (11/12 free-ranging, 5/6 wild-caught, captive, and 1/2 captive-bred). One captive-bred animal that died following an acute illness was positive on PCR only for MaHV4, an alphaherpesvirus previously identified from an eastern grey kangaroo (Macropus giganteus). The detection of MaHV4, associated with morbidity and mortality in captive tree-kangaroos, raises biosecurity concerns about introducing a non-endemic alphaherpesvirus into naive wild populations through release of captive animals. We propose that: 1) further work on herpesviruses in marsupials be carried out to determine whether herpesviruses from captive individuals represent a potential threat to wild populations, particularly for endangered species in which there are captive breeding and cross-fostering programs; and 2) that captive tree kangaroos be kept in such a way that prevents cross-species transmission of herpesviruses, in particular eliminating close direct or indirect contact with other species of macropods. © Wildlife Disease Association 2020.