Right ventricular (RV) strain values by 2-dimensional strain echocardiography (STE) can be used as objective markers of RV systolic function. However, there is little data about normal reference RV strain values according to age and gender. We measured normal RV strain values by STE. RV strain values were analyzed from the digitally stored echocardiographic images from NORMAL (Normal echOcardiogRaphic diMensions and functions in KoreAn popuLation) study for the measurement of normal echocardiographic values performed in 23 Korean university hospitals. We enrolled total 1003 healthy persons in the NORMAL study. Of them, we analyzed 2-dimensional RV strain values in 493 subjects (261 females, mean 47 ± 15 years old) only with echocardiographic images by GE machines. Their LV systolic and diastolic functions were normal. RV fractional area change was 48 ± 6% and tricuspid annular plane systolic excursion was 23 ± 3 mm. Total RV global longitudinal peak systolic strain (RVGLS_total) was −21.5 ± 3.2%. Females had higher absolute RVGLS_total (−22.3 ± 3.3 vs −20.7 ± 2.9%, p < 0.001) than males. Younger (<50 years old) females had higher absolute RVGLS_total (−22.9 ± 3.2 vs −20.5 ± 2.8%, p < 0.001) than age matched males. RVGLS_total in females gradually increased according to age (p for trend = 0.002) and becomes almost similar in age ≥50 years. However, this trend was not seen in males (p for trend = 0.287), and younger males had similar RVGLS_total value to that of older males (age ≥50 years, −20.5 ± 2.8 vs −20.9 ± 3.1%, p = 0.224). We calculated normal RVGLS values in normal population. Females have higher absolute strain values than males, especially in younger age groups (<50 years old).