Abstract Before in vivo micro-CT scanning can be used to investigate femoral trabecular microarchitecture over time in rabbits, its repeatability and reproducibility must be demonstrated. To accomplish this, both distal femurs of two 6-month-old New Zealand white rabbits were scanned five times each in 1 day under different conditions (repeatability). Scanning was done at 28 μm isotropic voxel size to produce five image stacks of each femur. Three operators then followed a standard image processing protocol (reproducibility) to isolate two separate cubes from each anterior femoral condyle [total n=(8 cube sites)(5 scans)(3 operators)=120]. Bone volume fraction (BV/TV) of the eight different cube sites (sample) ranged from 0.408 to 0.501 (mean: 0.453); trabecular thickness (Tb.Th) ranged from 158.1 to 185.5 μm (mean: 168.6 μm); and trabecular separation (Tb.Sp) ranged from 179.4 to 233.1 μm (mean: 204.7 μm). Using ANOVA and the variance component method, the total process variation was ±14.1% of the mean BV/TV of 0.453. The sample variation was ±13.9% ( p<0.001), the repeatability was ±2.1% ( p<0.001), and the reproducibility was ±0.1% ( p>0.05). Results were similar for Tb.Th and Tb.Sp. Though the contribution due to repeatability was statistically significant for each of the three indices, the natural sample differences were far greater than differences caused by repeated scanning under different conditions or by different operators processing the images. These findings suggest that in vivo micro-CT scanning of rabbit distal femurs was repeatable and reproducible and can be used with confidence to measure differences in trabecular bone microarchitecture at a single location in a longitudinal study design.