A growing concern that overshadows the increased life expectancy developed countries have been witnessing during the last decades is an accompanying bone loss, which often manifests as osteoporosis. Despite ongoing efforts in utilizing genomic data to fully map the genes responsible for bone remodeling, a detailed picture remains to be desired. Here we took advantage of the phenotypic and genetic diversity innate in Collaborative Cross (CC) mice to identify genetic variants associated with microstructural bone characteristics. We gauged several key femoral microarchitecture features of the femoral bone: bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), structural morphometric index (SMI) and spacing (Tb.Sp) of the trabecula, and thickness (Ct.Th) and volumetric bone mineral density (vBMD) of the cortex, to uncover possible genes by which these might be affected. We found 5 loci associated with 6 of the traits – BV/TV, Tb.N (one mutual locus), Tb.Th, Tb.Sp, vBMD, and Ct.Th (one locus for each). The broad-sense heritability of these traits ranged between 50 to 60%. The cortical traits were especially sensitive to confounders, such as batch, month and season. We refined each locus by combining information mined from existing databases with that obtained from the known ancestry of the mice, to shortlist potential candidate genes. We found strong evidence for new candidate genes, in particular, Rhbdf2. Using Rhbdf2 knockout mice, we confirmed its strong influence on bone microarchitecture; Rhbdf2-/- mice exhibit strikingly heightened BV/TV and Tb.N, heightened SMI and Ct.Th, lowered Conn.D and Tb.Sp, while Tb.Th and vBMD were unaffected, compared with wild type mice. High-resolution bone microarchitecture imaging of the CC population enabled us to further dissect the genetic makeup of bone morphology, and demonstrate conclusively that Rhbdf2 reduces the number and volume of the femoral trabecula.