Multilayer strain measurement with cardiac magnetic resonance feature tracking (CMR-FT) allows independent assessment of endocardial and epicardial strain. This novel method of layer-specific quantification of myocardial deformation parameters provides greater insight into contractility compared to whole-layer strain analysis. The clinical utility of this technique is promising. The aim of this study is to investigate the intra- and inter- observer reproducibility of CMR-FT derived multilayer global longitudinal strain (GLS) and global circumferential strain (GCS) parameters in the setting of normal cardiac function, cardiac pathology, and differing MRI field strengths. We studied 4 groups of 20 subjects, comprising of patients with dilated cardiomyopathy, ischemic heart disease, and patients without cardiac pathology at both 1.5 and 3 T. Quantitative measures of whole-layer and multi-layer longitudinal and circumferential strain were calculated using CMR-FT software. Intraclass correlation coefficients (ICC) for intraobserver reproducibility of endocardial, epicardial, and whole-layer measurements of GLS were 0.979, 0.980, and 0.978 respectively, and those for GCS were 0.986, 0.977, and 0.985. ICCs for inter-observer reproducibility of endocardial, epicardial, and whole-layer measurements of GLS were 0.976, 0.970, and 0.976, and those for GCS were 0.982, 0.969, and 0.981. Bland Altman analysis showed minimal bias and acceptable limits of agreement (LOA) within each patient subgroup and the overall cohort. Circumferential and longitudinal strain parameters were equally reproducible in the overall cohort. CMR-FT derived multilayer measurements of longitudinal and circumferential strain demonstrate high intra- and inter- observer reproducibility, with suitability for use in clinical practice. 2020 Cardiovascular Diagnosis and Therapy. All rights reserved.