The detailed anatomy of the interatrial septum (IAS) and mitral annulus (MA) as observed on cardiac magnetic resonance, computed tomography and two-dimensional/three-dimensional transthoracic and transesophageal echocardiography is reviewed. The IAS comprises of two components: the septum primum that is membrane-like forming the floor of the fossa ovalis (FO) and the septum secundum that is a muscular rim that surrounds the FO. The latter is an enfolding of atrial wall forming an interatrial groove. Named Waterston's groove, it is filled with adipose tissue on the epicardial side. Thus, the safest area for transseptal puncture (TSP) is within the limits of the FO floor, which provides direct interatrial access. While crossing an intact septum is a well-established procedure, TSP is a more complex and time-consuming procedure in the presence of patent foramen ovalis, aneurysmal FO or atrial septal defect closure devices. MA comprises two distinctive segments: an anterior-straight and a posterior-curved segment. The posterior MA is a thin, discontinuous fibrous 'string', interspersed with adipose tissue, where four components converge: the atrial and ventricular musculature, epicardial adipose tissue and the leaflet's hinge line. In parts of where this fibrous string is deficient or absent, the posterior leaflet is inserted directly on ventricular and atrial myocardium rendering the MA less robust and producing an 'asymmetric' dilation. The marked vulnerability of posterior MA to calcifications might be due to its insertion on the crest of ventricular myocardium being subject to friction injury due to the contraction and relaxation of LV.