Use of nanoindentation technology to identify quantitative trait loci (QTL) that regulate bone properties represents a novel approach to improving our understanding of molecular mechanisms that control bone matrix properties. Tibiae for QTL mapping were from an F2 population derived from C57BL/6J and C3H/HeJ. A nanoindenter (Triboindenter; Hysitron, Minneapolis, MN) was used to conduct indentation tests on transverse sections. Genotyping was performed in The Jackson Laboratory. QTL mapping was conducted using software. We found that (1) tibiae from mice at 16 weeks of age were mature and suitable for measurement by a nanoindentor; (2) both stiffness modulus and hardness modulus in the F2 population appeared to have normal distributions, which suggested that multiple genetic factors control the bone properties; and (3) QTL for hardness were identified from five chromosomes (Chr 8, 12, 13, 17, and 19) and for stiffness, from four chromosomes (Chr 3, 8, 12, and 13). Among all detected QTL, one at the same location on Chr 12 was detected for both hardness and stiffness data. It explained the highest percentage of phenotypic variation in bone properties. Using nanoindentation technology to identify QTL that regulate bone properties yielded as many as six different chromosomal regions. Although the actual genes remain to be identified, nanoindentation will contribute to our understanding of molecular mechanisms and normal development processes that control the matrix properties of bone.