Medical evaluations were performed on free-ranging and captive Matschie's tree kangaroos (Dendrolagus matschiei) in Papua New Guinea. The health assessment included physical examination, morphometrics, cloacal swab; and blood, hair, and feces collection. Radio-collars were placed on free-ranging tree kangaroos to determine home range and forest habitat use. The free-ranging tree kangaroos were lightly anesthetized with tiletamine/zolazepam for the data collection. A total of nine free-ranging and seven captive tree kangaroos were evaluated; medical samples were collected from six and five animals, respectively. Results of physical examination, anesthetic monitoring, serum vitamin, mineral, trace nutrient, and electrolytes, whole blood heavy metal analysis, mycobacterial screening, and fecal examinations are presented. Free-ranging tree kangaroos had significantly lower values for beta carotene, copper, selenium, molybdenum, lead, and arsenic and significantly higher values for vitamin E than captive individuals. Cloacal swabs were all negative for Mycobacterium avium via polymerase chain reaction. Some free-ranging and captive individuals had positive coprologic exams revealing Eimeria spp. oocysts and strongyle spp. type ova. These are the first medical and anesthetic data published on Matschie's tree kangaroos from Papua New Guinea.