Immobilization causes rapid and massive bone loss. By comparing Botulinum Toxin A (BTX)-induced bone loss in mouse strains with different genetic backgrounds we investigated whether the genetic background had an influence on the severity of the osteopenia. Secondly, we investigated whether BTX had systemic effects on bone. Female mice from four inbred mouse strains (BALB/cJ, C57BL/6J, DBA/2J, and C3H/HeN) were injected unilaterally with BTX (n = 10/group) or unilaterally with saline (n = 10/group). Mice were euthanized after 21 days, and the bone properties evaluated using μCT, DXA, bone histomorphometry, and mechanical testing. BTX resulted in substantially lower trabecular bone volume fraction (BV/TV) and trabecular thickness in all mouse strains. The deterioration of BV/TV was significantly greater in C57BL/6J (− 57%) and DBA/2J (− 60%) than in BALB/cJ (− 45%) and C3H/HeN (− 34%) mice. The loss of femoral neck fracture strength was significantly greater in C57BL/6J (− 47%) and DBA/2J (− 45%) than in C3H (− 25%) mice and likewise the loss of mid-femoral fracture strength was greater in C57BL/6J (− 17%), DBA/2J (− 12%), and BALB/cJ (− 9%) than in C3H/HeN (− 1%) mice, which were unaffected. Using high resolution μCT we found no evidence of a systemic effect on any of the microstructural parameters of the contralateral limb. Likewise, there was no evidence of a systemic effect on the bone strength in any mouse strain. We did, however, find a small systemic effect on aBMD in DBA/2J and C3H/HeN mice. The present study shows that BTX-induced immobilization causes the greatest loss of cortical and trabecular bone in C57BL/6J and DBA/2J mice. A smaller loss of bone microstructure and fracture strength was seen in BALB/cJ mice, while the bone microstructure and fracture strength of C3H/HeN mice were markedly less affected. This indicates that BTX-induced loss of bone is mouse strain dependent. We found only minimal systemic effects of BTX.