Objective: The objective was to evaluate intrarater and inter-rater reliability of ultrasonography muscle morphology measurements in men and women, considering morphological differences between the genders. Materials and Methods: Thirty-two healthy subjects (16 male; 16 female; 26.6 ± 4.9 years) participated in two evaluation days. On day 1, subjects were evaluated by a single experienced rater, who repeated the evaluation on the next testing day along with two other raters. Muscle morphology of quadriceps femoris (QMT), rectus femoris (RFMT), vastus intermedius (VIMT), vastus medialis (VMMT), and vastus lateralis (VLMT) muscle thickness, rectus femoris muscle cross-sectional area (RFCSA), vastus lateralis muscle fascicle length (VLFL), and pennation angle (VLPA) were obtained. Reliability was evaluated by intraclass correlation coefficients (ICCs). Results: All intrarater comparisons demonstrated good reliability (ICC >0.90), even after participants’ gender stratification (ICC >0.81). Inter-rater comparisons for RFCSA, and muscle thickness showed good reliability (ICC >0.76). Male’s VLFL and VLPA reliability was considered insufficient (ICC = 0.58 and 0.60, respectively), while female’s was slightly higher (ICC = 0.79 and 0.75, respectively). Conclusion: Ultrasonography has potential to be used to observe changes in muscle size, but pennation angle and fascicle length should be evaluated by the same rater.