Trabecular bone (Tb) is used for rapid exchange of calcium (Ca) in times of physiologic need and the site-specific characteristics of Tb may explain why certain sites are more vulnerable to osteoporosis. We hypothesized that peak trabecular bone mass (PTBM) and Tb microarchitecture are differentially regulated by dietary Ca intake, genetics, or Gene-by-Diet (GxD) interactions at the distal femur and the fifth lumbar (L5) vertebra. Male mice from 62 genetically distinct lines were fed basal (0.5%) or low (0.25%) Ca diets from 4 to 12 wks of age. Afterwards, the right femur and L5 vertebra were removed and trabecular bone was analyzed by μCT. In mice fed the basal diet, bone volume fraction (BV/TV), trabecular number (Tb.N), and connectivity density (Conn.D) were significantly higher in the L5 vertebra than femur. Femur Tb had a weaker, more rod-like structure than the L5 vertebrae while mice fed the low Ca diet developed rod-like structures at both sites. Dietary Ca restriction also caused a greater relative reduction of Tb.N and Conn.D in the femur than L5 vertebra, i.e. it was more harmful to the integrity of Tb microarchitecture in femur. Genetics was a major determinant of Tb at both sites, e.g. heritability of BV/TV on the basal diet = 0.65 (femur) and 0.68 (L5 vertebra). However, while GxD interactions altered the impact of dietary Ca restriction on Tb parameters at both sites, the effect was not uniform, e.g. some lines had site-specific responses to Ca restriction. The significance of our work is that there are site-specific effects of dietary Ca restriction and genetics that work independently and interactively to influence the attainment of PTBM and Tb microarchitecture.