Introduction: Echocardiographic evaluation of 2D longitudinal peak systolic strain (LPSS) can detect initial impairment of left ventricular (LV) function in heart disease. Global LPSS (GLPSS) variability has been assessed in small groups and segmental LPSS has not been determined. We compared variability of GLPSS and segmental LPSS with that of 2D LV volumes and ejection fraction (EF) in patients with and without heart diseases. Methods: 2D speckle tracking analysis was performed on LV apical views using AFI soft ware (GE Healthcare, v112). Intraoperator, intercycle and test – retest variability (bias and CR, coeffi cient of reproducibility; MPE, mean percent error; CV, coeffi cient of variation) was assessed for GLPSS, 18 segments of LPSS, and LV volumes and EF in 40 patients (740 segments), and interoperator variability in 250 patients (4500 segments). Results: Feasibility of segmental tracking was 92%. Variability of GLPSS increased from a minimum intraoperator CV = -2.6%, to a maximum test – retest CV = -3.8% and was (better) lower than that assessed for LV volumes and EF. Segmental intra-operator LPSS CV ranged from -5.6% to -14.7%, and test – retest from -8% to -22% and was at worst similar to variability of end-systolic volume. In the 8.3% of segments with the highest variability, this was related to suboptimal imaging, minor changes in scan angulation and insufficient ROI width. Conclusions: Overall reproducibility of GLPSS is excellent and superior to that of 2D EF, whereas segmental LPSS reproducibility is good and similar to that of LV volumes. Both are suitable for diagnosis and followup of LV global and regional systolic function.