We aimed to establish a novel approach with 3D high frequency power Doppler ultrasonography (3D-HF-PDU) to assess microvasculature at the fracture site in rat femurs by comparing with microCT-based microangiography. Twenty-four 9-month-old ovariectomized (OVX) osteoporotic rats and age-matched sham-ovariectomized (Sham) rats were used for establishing closed fracture models on right femora. At 2, 4, and 8 weeks post-operatively, four rats in each group underwent in vivo 3D-HF-PDU scanning for evaluation of vascularization and blood flow at the fracture site. Then the fractured femora were harvested for ex vivo microangiography, and neovasculatures within the callus were reconstructed for vascular volume analysis. Correlation between the vascular volumes of the two methodologies was examined. Both 3D-HF-PDU and microangiography showed a decline of vascular volume at the fracture site from 2 to 8 weeks and a significantly larger volume in the Sham group than the OVX group. A significant linear positive correlation (r = 0.87, p < 0.001) was detected between the volumes measured by the two methodologies. Osteoporotic rats had a diminished angiogenic response and lower blood perfusion than Shams. We believe 3D-HF-PDU is feasible and reproducible for in vivo assessment of microvasculature during femoral fracture healing in rats.