During disuse, mechanical unloading causes extensive bone loss, decreasing bone volume and strength. Variations in bone mass and risk of osteoporosis are influenced by genetics; however, it remains unclear how genetic variation affects the skeletal response to unloading. We previously found that genetic variation affects the musculoskeletal response to 3 weeks of immobilization in the 8 Jackson Laboratory J:DO founder strains: C57Bl/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Hindlimb unloading (HLU) is the best model for simulating local and systemic contributors of disuse and therefore may have a greater impact on bones than immobilization. We hypothesized that genetic variation would affect the response to HLU across the eight founder strains. Mice of each founder strain were placed in HLU for 3 weeks, and the femurs and tibias were analyzed. There were significant HLU and mouse strain interactions on body weight, femur trabecular BV/TV, and femur ultimate force. This indicates that unloading only caused significant catabolic effects in some mouse strains. C57BL/6 J mice were most affected by unloading while other strains were more protected. There were significant HLU and mouse strain interactions on gene expression of genes encoding bone metabolism genes in the tibia. This indicates that unloading only caused significant effects on bone metabolism genes in some mouse strains. Different mouse strains respond to HLU differently, and this can be explained by genetic differences. These results suggest the outbred J:DO mice will be a powerful model for examining the effects of genetics on the skeletal response to HLU.