Vastus medialis (VM) has two partitions, longus (VML), and obliquus (VMO), which have been implicated in knee pathologies. However, muscle architecture of VMO and VML has not been documented volumetrically. The aims of this study were to determine and compare the muscle architecture of VMO and VML in three-dimensional (3D) space, and to elucidate their relative functional capabilities. Twelve embalmed specimens were used in this study. Each specimen was serially dissected, digitized (Microscribe™ MX), and modeled in 3D (Autodesk Maya®). Architectural parameters: fiber bundle length (FBL), proximal (PPA)/distal (DPA) pennation angle, and physiological cross-sectional area (PCSA) were compared using descriptive statistics/t-tests. Sarcomere lengths (SLs) were measured and compared from six biopsy sites of VM. VMO and VML were found to have superficial and deep parts based on fiber bundle attachments to aponeuroses, medial patellar retinaculum, and adductor magnus tendon. The superficial part of VMO was further subdivided into superior and inferior partitions. Architecturally, VMO was found to have significantly shorter mean FBL, greater mean PPA and DPA, and smaller mean PCSA than VML. VML was found to be connected to the fascia lata by thin fascial bands, not present in VMO. SLs of VMO and VML were comparable. VMO and VML are architecturally and functionally distinct, as evidenced by marked differences in their musculoaponeurotic geometry, attachment sites, and architectural parameters. VMO likely contributes greater to medial patellar stabilization, whereas VML, with a larger relative excursion and force-generating capability, to the extension of the knee. Clin. Anat. 32:515-523, 2019. © 2019 Wiley Periodicals, Inc.