INTRODUCTION: The assessment of left ventricular myocardial deformation and function by two-dimensional speckle tracking-derived strain analysis is an established method in human cardiology. It also progressively gains recognition in veterinary cardiology in both dogs and cats. OBJECTIVES: The objectives of this study were to create reference intervals for two-dimensional speckle tracking echocardiography (STE)-derived endocardial global longitudinal strain (GLS) in a population of healthy adult cats of different breeds. Influences of heart rate, body weight, and age were investigated. ANIMALS: A total of 132 healthy, adult cats were included in this study. MATERIALS AND METHODS: Left apical two-, three-, and four-chamber views were obtained prospectively for GLS measurements using two-dimensional speckle tracking performed with cardiac performance analysis. Potential influence of body weight, heart rate, and age was analyzed, and the interobserver and intra-observer variability of the measurements was determined. RESULTS: Endocardial GLS values were not significantly influenced by body weight (P=0.102), heart rate (P=0.144), or age (P=0.075). A reference interval for GLS of -21.18% to -37.50% (±4.12) was determined. The interobserver and intra-observer variability showed excellent agreement. DISCUSSION AND CONCLUSIONS: Two-dimensional STE is a feasible technique for the evaluation of cardiac myocardial deformation and systolic function in cats. Showing an excellent interobserver and intra-observer agreement, two-dimensional STE is a promising method for clinical analysis of cardiac deformation in cats.