Dual-energy X-ray absorptiometry (DXA) is a non-invasive technology for measurement of body composition that requires validation against reference methods when applied to a new species. The aim of this work was to validate DXA for the assessment of body composition of the echidna. Body composition was determined in the short-beaked echidna (Tachyglossus aculeatus aculeatus) using a Norland XR36 DXA scanner and validated by proximate chemical analysis for dry matter, ash, crude fat (FM) and protein (as 6.25 × N) and bone mineral content (BMC). Echidnas were opportunistically obtained as ‘road kill’. Body composition data were compared between techniques by correlation and limits of agreement (LOA) analyses. Twenty-eight echidnas (11 males, 13 females, 4 not determined), weighing 520–5517 g, underwent analyses. Mean FM was 489.9 ± 439.5 g and 448.5 ± 337.5 g, lean mass was 2276.0 ± 1021.4 g and 2256.0 ± 1026.0 g, fat-free mass was 2356.3 ± 1055.1 g and 2389.5 ± 1081.1 g and BMC was 80.3 ± 39.5 g and 79.9 ± 42.4 g by DXA and chemical analysis, respectively. The two methods were highly correlated (0.84 to 0.99) and not significantly different, although LOA were large. DXA has the potential to be used to assess body composition of echidnas although further work is required to improve accuracy of measurement.