This systematic review aimed to evaluate the literature on the acquisition-, reconstruction- and analysis parameters of micro-computed tomography (micro-CT) for the assessment of periapical lesions in rats and mice, and to illustrate the effect of variation in these parameters. The PubMed database was searched from 2000 to January 2015 (English-language publications) for reports on the use of micro-CT to evaluate periapical lesions in rats and mice. QUADAS criteria were used to rate the quality of the studies. To illustrate the effect of variations in acquisition-, reconstruction-, and analysis parameters on images of periapical lesions, micro-CT examination of two hemi-mandibles of mice, with periapical lesions around the first molar was undertaken. Twenty-one studies were identified, which analysed periapical lesions in rats or mice using micro-CT. According to the QUADAS, no study was classified as high-, seven were classified as moderate-, and 14 as low quality. The effect of variation in parameters was that voxel size may interfere with image sharpness, reconstruction may interfere with image sharpness and contrast, and inadequate plane orientation may alter the size of the periapical lesion. Nonpersonalized ROIs resulted in areas that were not part of the periapical lesion. Changing the limits of the threshold for bone-tissue visualization increased lesion size. There is no defined protocol for acquiring and analysing micro-CT images of periapical lesions in rats and mice. Furthermore, acquisition-, reconstruction- and analysis parameters are not adequately explained, which may compromise the scientific impact of the studies.