We recently showed that Quantitative Perfusion SPECT (QPS), Emory Cardiac Toolbox (ECTb) and 4-Dimension-Myocardial SPECT (4DM) yielded discordant results in the assessment of perfusion defect size (PDS) in patients with known or suspected coronary artery disease. In this report, we examined the sources of variability in the measurement of PDS using ECTb (VarECTb), 4DM (Var4DM) and QPS (VarQPS) and found that gender is a source of variability in the assessment of PDS. In 120 patients, VarECTb was lower in men, compared with women, whereas Var4DM and VarQPS were similar between both genders. Among men, VarECTb was lowest, compared with VarQPS and Var4DM, whereas, VarECTb, VarQPS, and Var4DM were not different among women. Future studies should account for gender differences when comparing the 3 software programs.