Background: Backscatter difference techniques are being developed to detect changes in bone caused by osteoporosis. Backscatter difference techniques compare the power in one portion of an ultrasonic backscatter signal to the power in a different portion of the same signal. Goal: Investigate how backscatter difference measurements depend on the density and microstructural characteristics of cancellous bone. Procedure: Ultrasonic backscatter signals were acquired from 30 specimens of bone using a 1 and 5 MHz broadband transducer. The normalized mean backscatter difference (nMBD) was determined by computing the power difference (in dB) between two gated portions of the backscatter signal and dividing by the center to center time separation between gates. Microstructural characteristics of the specimens and bone mineral density (BMD) were determined using high resolution x-ray micro-computed tomography. Results: nMBD demonstrated moderate to strong linear correlations with microstructure and BMD (0.50 ≤ |R| ≤ 0.83). The measured correlations did not depend strongly on transducer frequency. Conclusions: The backscatter difference parameter nMBD may be sensitive to changes in microstructure and density caused by osteoporosis. [Work supported by NIH/NIAMS R15AR066900.]