MicroCT-based morphological parameters are often used to quantify the structural properties of trabecular bone. Various software tools are available for calculating these parameters. Studies that examine the comparability of their results are rare. Four different software tools were used to analyse a set of 701 microCT images from human trabecular bone samples. Bone volume to total volume (BV/TV), bone surface (BS), trabecular thickness (Tb. Th.) and degree of anisotropy (DA) were evaluated. BV/TV shows very low difference (−0.18 ± 0.15%). The difference in BS could be reduced below 5% if artificial cut surfaces are not included. Tb. Th. and Tb. Sp. show differences of maximal −12% although the same theoretical background is used. DA is most critical with differences from 4.75 ± 3.70% (medtool vs. Scanco), over −38.61 ± 13.15% (BoneJ vs. Scanco), up to 80.52 ± 50.04% (medtool vs. BoneJ). Quantitative results should be considered with caution, especially when comparing different studies. Introducing standardization procedures and the disclosure of underlying algorithms and their respective implementations could improve this issue.