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PurposeTwo-dimensional (2D) and three-dimensional (3D) speckle-tracking echocardiography (STE) enable objective assessment of myocardial function. Here we examined the agreement between 2D and 3D STE measurement of global longitudinal strain (GLS) in patients with normal left ventricle, reduced ejection fraction, and cardiac pacing. MethodsOur analysis included 90 consecutive patients (59% males; average age: 73.2 ± 11.2 years) examined between May 2019–December 2020, with valid 2D and 3D loops for further speckle-tracking strain analysis. Linear regression and Pearson correlation were used to quantify the association between 2D and 3D GLS, and related segments using the 17-segment American Heart Association (AHA) model. Analyses were performed in the entire study group and subgroups. ResultsWe observed a strong correlation between 2D and 3D GLS measurements (R = 0.76, P < 0.001), which was higher in males (R = 0.78, P < 0.001) than females (R = 0.69, P < 0.001). Associated segment correlation was poor (R = 0.2–0.5, P < 0.01). The correlation between 2D and 3D GLS was weaker in individuals with ventricular pacing of >50% (R = 0.62, P < 0.001) than <50% (R = 0.8, P < 0.001), and in patients with LVEF of <35% (R = 0.69, P = 0.002) than >35% (R = 0.72, P <0.001). Conclusionverall 2D and 3D GLS were closely associated, but not when analyzed per segment. Right ventricular pacing and reduced left ventricular ejection fraction were associated with reduced correlation between 2D and 3D GLS.