The aim of this study was to compare the performance of three different software packages for the calculation of ejection fraction (EF) and end diastolic volume (EDV) from gated myocardial single photon emission computed tomography studies. Two hundred patients undergoing gated stress myocardial perfusion scans were analysed retrospectively. Patients were grouped as follows: small heart (n = 31), normal perfusion scan (n = 71), and scan with perfusion defects (n = 98). EF and EDV were calculated for each using QGS (Cedars Sinai, Los Angeles, CA), 4D-MSPECT (University of Michigan, Ann Arbor, MI), and ECT (Emory University, Atlanta, GA). Bland-Altman plots, repeated measures ANOVA, and linear regression analysis were used to compare methods. Correlation coefficients between the methods for both EF and EDV were high, greater than 0.9. However, Bland-Altman plots revealed a large standard deviation of the difference between methods, preventing the confident estimate of the value of one method from an observation of another. Despite good correlation, the variance between methods was high. These algorithms behave differently, produce widely variable results from one another, and should not be used interchangeably. It may prove prudent for laboratories to independently validate the software algorithm that is chosen against a ‘gold standard’ using their own population.