Background Conventional transthoracic (TTE) and transoesophageal echocardiography (TEE) parameters assessing right ventricle (RV) systolic function are daily used assuming their clinical interchangeability. RV longitudinal shortening fraction (RV-LSF) is a two-dimensional speckle tracking parameter used to assess RV systolic function. RV-LSF is based on tricuspid annular displacement analysis and could be measured with TTE or TEE. Objective The aim of the study was to determine if RV-LSF TTE and RV-LSF TEE measurements were interchangeable in the perioperative setting. Methods Prospective perioperative TTE and TEE echocardiography were performed under general anesthesia during scheduled cardiac surgery in 90 patients. RV-LSF was measured by semi-automatic software. Comparisons were performed using Pearson correlation and Bland-Altman plots. RV-LSF clinical agreement was determined as a range of −5 to 5%. Results Of the 114 patients who met the inclusion criteria, 90 were included. The mean preoperative RV-LSF TTE was 20.4 ± 4.3 and 21.1 ± 4.1% for RV-LSF TEE. The agreement between RV-LSF measurements was excellent, with a bias at −0.61 and limits of agreement of −4.18 to 2.97 %. All measurements fell within the determined clinical agreement interval in the Bland-Altman plot. Linear regression analysis showed a high correlation between RV-LSF TTE and RV-LSF TEE measurement (r = 0.9; confidence interval [CI] 95%: [0.87–0.94], p < 0.001). Conclusion RV-LSF TTE and RV-LSF TEE measurements are interchangeable, allowing RV-LSF to be a helpful parameter for assessing perioperative changes in RV systolic function. NCT NCT05404737. https://www.clinicaltrials.gov/ct2/show/NCT05404737 .