In the clinical environment dual-energy X-ray absorptiometry (DXA) is the current tool of first choice for assessing and monitoring skeletal integrity. A major drawback of standard DXA is that the bone mineral density (BMD) data cannot be used with certainty to predict who will sustain a vertebral fracture. However, measurement of BMD within vertebral subregions, instead of relying on a gross estimate of vertebral BMD, may improve diagnostic sensitivity. The aim of this article was to describe a validation study for subregional BMD measurement using lateral-projection DXA and to present preliminary data. Concurrent validity of measuring subregional BMD with DXA was established against measures of volumetric subregional BMD from peripheral quantitative computed tomography (pQCT) and subregional bone volume fraction from μCT at the L2 vertebral body in 8 cadaver spine specimens. The novel approaches for measuring subregional parameters with each imaging modality are described. Significant differences in bone parameters between vertebral subregions were observed for each imaging modality (p < 0.05). Correspondence ranged from R2 = 0.01-0.79 and R2 = 0.06-0.80 between "DXA vs. pQCT" and "DXA vs. micro-CT," respectively. For both imaging modalities, correspondence with DXA was high for centrally and anteriorly positioned subregions. These data provide a basis for larger studies to examine the biological significance of heterogeneity in vertebral BMD.