Background Masseter muscle function influences mandibular bone homeostasis. As previously reported, bone resorption markers increased in the mouse mandibular condyle two days after masseter paralysis induced with botulinum toxin type A (BoNTA), followed by local bone loss. Objective This study aimed to evaluate the bone quality of both the mandibular condyle and alveolar process in the mandible of adult mice during the early stage of a BoNTA-induced masseter muscle atrophy, using a combined 3D histomorphometrics and shape analysis approach. Methods Adult BALB/c mice were divided into an untreated control group and an experimental group; the latter received one single BoNTA injection in the right masseter (BoNTA-right) and saline in the left masseter (Saline-left). 3D bone microstructural changes in the mandibular condyle and alveolar process were determined with high-resolution microtomography. Additionally, landmark-based geometric morphometrics was implemented to assess external shape changes. Results After 2 weeks, masseter mass was significantly reduced (P-value <0.001). When compared to Saline-left and untreated condyles, BoNTA-right condyles showed significant bone loss (P-value <0.001) and shape changes. No significant bone loss was observed in the alveolar processes of any of the groups (P-value >0.05). Conclusion Condyle bone quality deteriorates at an early stage of BoNTA-induced masseter muscle atrophy, and before the alveolar process is affected. Since the observed bone microstructural changes resemble those in human temporomandibular joint degenerative disorders, the clinical safety of BoNTA intervention in the masticatory apparatus remains to be clarified.