Aim: To promote the implementation of right ventricular (RV) longitudinal strain in clinical practice, we sought to propose normal values for RV free wall (RVFWLS) and four-chamber longitudinal strain (RV4CLS) and investigate the association with clinical and echocardiographic parameters in participants from the general population. Methods and Results: Participants from the 5th Copenhagen City Heart Study (2011–2015)—a prospective cohort study—with available RV longitudinal strain measurements were included. RVFWLS and RV4CLS were assessed using two-dimensional speckle-tracking echocardiography. In total, 2951 participants were included. Amongst 1297 participants without cardiovascular disease or risk factors (median age 44, 63% female), mean values of RVFWLS and RV4CLS were − 26.7% ± 5.2 (95% prediction interval (PI) − 36.9, − 16.5) and − 21.7% ± 3.4 (95%PI − 28.4, − 15.0), respectively. Women had significantly higher absolute values of RVFWLS and RV4CLS than men (mean − 27.5 ± 5.5 vs. − 25.4 ± 4.5, p < 0.001 and − 22.3 ± 3.5 vs. − 20.6 ± 3.0, p < 0.001, respectively). Absolute values of RVFWLS but not RV4CLS decreased significantly with increasing age in unadjusted linear regression. Tricuspid annular plane systolic excursion, RV s’ and left ventricular global longitudinal strain were the most influential parameters associated with both RVFWLS and RV4CLS in multiple linear regression. Participants with cardiovascular disease (n = 1531) had a higher proportion of abnormal values of RVFWLS and RV4CLS compared to the healthy population (8% vs. 4%, p < 0.001 and 8% vs. 3%, p < 0.001, respectively). Conclusion: This study proposed normal age- and sex-based values of RVFWLS and RV4CLS in a healthy population sample and showed significant sex differences in both measurements across ages. Graphical abstract: [Figure not available: see fulltext.].