Speckle tracking echocardiography is an advanced imaging technique that allows for a more detailed assessment of cardiac global and regional function. Reference values for segmental longitudinal layered strain (subendocardial, mid-myocardial, and subepicardial) are scarce, limiting the clinical use of these measurements in clinical practice. Two hundred consecutive Caucasian healthy subjects (mean age = 37 ± 11 years) were enrolled in the study. The mean values of global longitudinal strain (GLS) for endocardial (Endo), mid-myocardial (Myo) and epicardial (Epi) layers were −22.9 ± 2.7, −20.0 ± 2.4 and −17.5 ± 2.1, respectively. The GLSEndo/GLSMyo ratio was 1.1 ± 0.05, while the GLSEndo/GLSEpi ratio was 1.3 ± 0.05. The apical strain-sparing ratio was >1 in 10% of the subjects (endocardium) and 7% (mid-myocardium). The lower limits for segmental LS were as follows: for endocardial LS, −10% (basal), −12% (mid), −14% (apical); for mid-myocardial LS, −10% −10% (basal), −10% (mid), −10% (apical); and for epicardial LS, −7% (basal), −8% (mid), −8% (apical). The findings of this study provide data regarding the lower limit of normality of LS for each LV segment and suggest, for practical considerations, that an LS value below 10% should be considered abnormal in any segment. Further larger studies are warranted to confirm these findings.