Trabeculae form the internal bony mesh work and provide strength to the bone; interconnectivity, overall density, and trabecular thickness are important measures of the integrity of the internal architecture. Such strength is achieved only gradually during ontogeny, whereby an increase in trabecular thickness precedes an increase in mineralization. Loss of bone mass later in life may be compensated for by thickening of the remaining trabeculae. These facts, and the role of trabeculae in mineral homeostasis, highlight the importance of investigating trabecular thickness within and between species. While nondestructive imaging techniques (i.e., μCT and MRI) are becoming increasingly popular, quantification of trabecular thickness using nondestructive techniques has proved difficult owing to limitations imposed by scanning parameters, uniform thresholding, and partial volume averaging. Here we present a computer application, which aims to overcome these problems. Validation is carried out against a phantom and against trabecular thickness measured in corresponding histological sections. Good agreement was found between these measurements. Furthermore, when trabecular thickness is recorded for modern human fetal ilia, a trend toward trabecular thickness increase is found and is in line with reports of ontogenetic morphometric changes using histological sections. However, there are discrepancies. These may in part be due to partial volume effects of obliquely oriented structures. More crucial, however, are problems inherent in histological sections, e.g., shrinkage and distortion, especially where differences in mineralization are concerned; this may affect biological interpretations. Anat Rec Part A, 288A:982–988, 2006. © 2006 Wiley-Liss, Inc.