Cardiomyocyte contraction is critically dependent on Ca2+ release from Ryanodine Receptors (RyRs). While advances in single-molecule localization microscopy have allowed 2D, nanoscale visualization of RyRs at the cell surface, little is known about the geometric arrangement of RyRs in the context of a whole cell. Here, we have developed a novel approach to quantitatively characterize the precise organization of RyRs in isolated adult Wister rat (male, 250-350 g) cardiomyocytes using 3D direct stochastic optical reconstruction microscopy (dSTORM) (Zeiss ELYRA). We observed that RyR cluster organization at the cell surface was undulatory, resulting in overlap in the z-axis which obfuscated detection by 2D techniques. Non-overlapping clusters were imaged to generate a calibration curve (3.3076 events/RyR, 1740 clusters) for estimating RyR number based on recorded fluorescent events. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially-aligned RyRs was circumvented (mean 3D cluster size= 10.1±1.1 and 13.1±1.0 RyRs at cell surface and interior, respectively).Functional groupings of RyR clusters localized within 100 nm (Ca2+ release units, CRUs) contained an average of 18 and 23 RyRs at the surface and interior. While smaller than previous reports, this estimate parallels RyR numbers calculated to underlie Ca2+ sparks. Half of all CRUs contained only a single “rogue” RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ≈75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex 3D geometry of internal CRUs was underscored by correlative imaging of RyRs and T-tubules, which enabled visualization and quantification of dyadic and non-dyadic RyR populations. These data provide novel, nanoscale insight into RyR organization in cardiomyocytes critical for understanding excitation-contraction coupling.