Classic echocardiographic methodologies offer limited opportunities in assessing right atrial (RA) morphology and function. Three-dimensional (3D) speckle-tracking echocardiography (3DSTE) is a novel imaging method with objective 3D capability in assessing volumetric and functional properties of heart chambers. Normal reference values of different 3DSTEderived RA strains are not available, therefore the aim of this prospective study was to establish these parameters in healthy subjects. The present study comprised 295 healthy volunteers, from which 110 were excluded due to inadequate image quality. The final population consisted of 185 healthy subjects in the present study (mean age: 32.1 ± 12.2 years, 89 males). Complete two-dimensional echocardiography and 3DSTE have been performed in all cases. While radial strain (RS) does not change significantly over the years in males, in female subjects it increases with age most significantly between at the age of 40-49, and it starts to decline at the age of 50 in females. While females have higher circumferential (CS) and area (AS) strain values, CS and AS decrease with age in both gender. While LS remains almost unchanged in females until ages 40-49 years with a decline above the age of 50, it decreases over the decades in males. 3D strain (3DS) increases with age in both gender, but almost doubles in females in older ages. Specific pattern of strains at atrial contraction could also be demonstrated. 3DSTE-derived RA normal reference values with age-, gender-dependency and regional values are demonstrated in a healthy population.