The C57BL/6 mouse, the most frequently utilized animal model in biomedical research, is in use as several substrains, all of which differ by a small array of genomic differences. Two of these substrains, C57BL/6J (B6J) and C57BL/6N (B6N), are commonly used but it is unclear how phenotypically similar or different they are. Here, we tested whether adolescent B6N mice have a bone phenotype and respond to the loss of weightbearing differently than B6J. At 9 weeks of age, normally ambulating B6N had lower trabecular bone volume fraction but greater bone formation rates and osteoblast surfaces than corresponding B6J. At 11 weeks of age, differences in trabecular indices persisted between the substrains but differences in cellular activity had ceased. Cortical bone indices were largely similar between the two substrains. Hindlimb unloading (HLU) induced similar degeneration of trabecular architecture and cellular activity in both substrains when comparing 11-week-old HLU mice to 11-week-old controls. However, unloaded B6N mice had smaller cortices than B6J. When comparing HLU to 9 weeks baseline control mice, deterioration in trabecular separation, osteoblast indices, and endocortical variables was significantly greater in B6N than B6J. These data indicate specific developmental differences in bone formation and morphology between B6N and B6J mice, giving rise to a differential response to mechanical unloading that may be modulated, in part, by the genes Herc2, Myo18b, and Acan. Our results emphasize that these substrains cannot be used interchangeably at least for investigations in which the phenotypic makeup and its response to extraneous stimuli are of interest.