We aimed to assess intra- and inter-observer reproducibility in evaluating volume and characteristics of non-calcified coronary plaques (NCPs) using a 256-slice multi-detector computed tomography (MDCT) angiography and dedicated automated plaque analysis software in asymptomatic individuals. Forty-two NCPs from 39 patients with a vessel diameter >2 mm were evaluated using a 256-slice MDCT with dedicated automated plaque analysis software. Two independent observers analyzed the characteristics of NCPs, including plaque volume (vol), mean CT number of the NCPs in Hounsfield units (HU_mean), and remodeling index (RI). One of the observers repeated the evaluation of all datasets after an interval of at least 4 weeks. Bland–Altman analysis and concordance correlation coefficients (CCCs) were used to determine intra- and inter-observer variability. For vol measurements, the 95% limits of agreement were −21.6 and 13.2 mm³, and −24.6 and 20.3 mm³ for intra- and inter-observer variability, respectively. For HU_mean measurements, the 95% limits of agreement were −22.2 and 20.8 HU, and −21.1 and 21.0 HU for intra- and inter-observer variability, respectively. For RI measurements, the 95% limits of agreement were −0.38 and 0.39, and −0.51 and 0.36 for intra- and inter-observer variability, respectively. The CCCs was very high for all measurements, ranging from 0.90 to 0.98. Using 256-slice MDCT with dedicated automated plaque analysis software, intra- and inter-observer reproducibility were excellent in evaluating the volume and characteristics of NCP in asymptomatic individuals.