To investigate microcracks as a potential index of bone biomechanical quality in rats, 98 Sprague-Dawley (SD) rats, 10 months old, were used. Eight rats were killed at the beginning of the study, to serve as baseline controls. The remaining 90 rats were ovariectomized (OVX), and treated with 17 Beta-estradiol (EST) at 10 microg/kg per day, or sham-operated (sham). These 90 rats were killed at 3, 15, and 12 weeks post-surgery. Bone mineral density (BMD) of the total body, the lumbar spine L1-L4, and the tibiae was measured, using dual-energy X-ray absorptiometry (DXA). Bone histomorphometry of the right proximal tibial metaphysis was performed. Compressive testing was performed on the L5 vertebral body. Microcrack density (CrDn) and microcrack surface density (CrSDn) of L4 vertebral bodies that were fatigue-damaged in vitro were determined from bone sections. BMD at various sites, and CrDn and CrSDn were higher at weeks 15 and 21 than at week 3 post-operation. Trabecular separation (TbSp) increased, while trabecular number (TbN) decreased, and the maximum loading (ML) and elastic modulus (EM) of the vertebrae reached their peak values at week 15. At week 3 post-surgery, OVX rats displayed greater TbSp, CrDn, and CrSDn but less trabecular bone volume (BV) than both sham and EST rats. At week 15, BMD and ML were decreased in OVX rats compared with sham rats. At week 21, BMD, TbN, and TbAr were decreased in OVX compared with EST and sham rats. The OVX rats showed greater TbSp, bone formation rate, mineral apposition rate, percent labeled perimeter (%Pm), CrDn, and CrSDn, and lower ML and EM values than both EST and sham rats. Thus, microcrack parameters represent bone biomechanical quality changes associated with ovariectomy in rats, and they indicate the efficacy of estrogen replacement therapy.