Muscle architecture parameters, such as the fascicle length, pennation angle, and volume, are important muscle morphology characteristics. Accurate in vivo quantification of these parameters allows to detect changes due to pathologies, interventions, and rehabilitation trainings, which ultimately impact on muscles’ force-producing capacity. In this study, we compared three-dimensional (3D) muscle architecture parameters of the tibialis anterior and gastrocnemius medialis, which were quantified by 3D freehand ultrasound (3DfUS) and a magnetic resonance imaging (MRI) technique, diffusion tensor imaging (DTI), respectively. Sixteen able-bodied subjects were recruited where seven of them received both 3DfUS and MRI measurement, while the rest underwent 3DfUS measurements twice. Good to excellent intra-rater reliability and inter-session repeatability were found in 3DfUS measurements (intra-class correlation coefficient > 0.81). Overall, the two imaging modalities yielded consistent measurements of the fascicle length, pennation angle, and volume with mean differences smaller than 2.9 mm, 1.8°, and 5.7 cm³, respectively. The only significant difference was found in the pennation angle of the tibialis anterior, although the discrepancy was small. Our study demonstrated, for the first time, that 3DfUS measurement had high reliability and repeatability for measurement of muscle architecture in vivo and could be regarded as an alternative to MRI for 3D evaluation of muscle morphology.