Listed as near-threatened by the International Union for Conservation of Nature (IUCN), the southern hairy-nosed wombat (SHNW, Lasiorhinus latifrons) faces threats such as drought, habitat degradation and loss, disease, and persecution because of competition with agriculture. To assist with evaluation of wombat health, this study reports serum biochemical reference intervals (RIs) for wild-caught SHNW from South Australia established from 126 apparently healthy SHNW using a Beckman Coulter AU480^® Automated Chemistry Analyzer using RefVal Advisor. Partitioning of RIs for male and female wombats and for the two methods of sampling was performed as appropriate, and additional significant differences (P < 0.05) in biochemical profiles were identified across age class and season examined. A number of differences were observed between male and female wombats, most notably higher creatinine, urea, and sodium in females. Subadult and juvenile wombats had significantly lower total protein, globulin, and ALT activity, and significantly higher ALP activity than adults. Wombats sampled in winter and spring had significantly higher total protein, albumin, potassium, bicarbonate, and enzyme activities (ALP, ALT, AST, GGT, GLDH, lipase), and significantly lower glucose and creatinine when compared to individuals sampled in summer and autumn. Differences in CK activity and anion gap observed between the two methods of sampling likely reflect delay and handling of animals between capture and blood collection. The serum biochemical RIs documented here are considered representative of a population of healthy SHNW, providing a tool for health assessment and monitoring of SHNW health in South Australia and elsewhere.