Although osteoporosis reduces overall bone mass causing bone fragility, recent studies report that the remaining bone tissue is significantly stiffer. Preliminary studies indicate that alterations in bone tissue mineral content might explain these changes, albeit that other studies report conflicting observations. The objective of this study is to quantify whether the distribution of bone tissue mineral is altered during oestrogen deficiency. Individual trabeculae were harvested from the proximal femur of 7 ovariectomised sheep (OVX), sacrificed 12 months post-surgery, and 5 age-matched controls. Mineral content (wt% Ca) was determined using a quantitative backscattered scanning electron microscopy imaging approach. Mineral heterogeneity within individual trabeculae was compared by calculating the full width at half maximum (FWHM) of mineral density distributions. Mean calcium content, the spatial distribution of mineral within trabeculae and the inter-trabecular variation between regions of proximal femora were also compared. Oestrogen deficiency increased mineral heterogeneity within individual trabeculae compared to healthy controls, as measured by FWHM (3.57 +/- 0.68 vs. 3.17 +/- 0.36 wt% Ca, p = 0.04). In particular mineral variability increased between superficial and deep regions of trabeculae of OVX animals (p = 0.04). Interestingly, mineralisation variability between greater and lesser trochanters (i.e. intertrochanteric fracture line) was increased in OVX compared to CON, as indicated by a greater % difference in the standard deviation of trabecular mineral content (77.11 +/- 11.70 vs. 45.64 +/- 23.70 %, p = 0.03). Such changes are undetectable by evaluating the mean mineral content of bone tissue, but may contribute to changes in bone mechanical strength following osteoporotic bone loss.