To assess the intra- and inter-observer repeatability of popular software packages for the quantitative determination of abnormality size in stress myocardial perfusion scintigraphy. A total of 182 tomographic stress myocardial perfusion scans were processed in duplicate by an experienced and trainee observer to assess SSSext (summed stress score multiplied by 100/68) and total defect extent (TDE), as % of the left ventricle, with 4 dimension-myocardial (4DM), emory cardiac toolbox (ECTb) and quantitative perfusion SPECT (QPS) packages. The Bland-Altman (B-A) analysis and Lin's concordance correlation coefficient (CCC) were used to assess agreement. In SSSext's intra-observer repeatability, CCC showed substantial agreement for 4DM and QPS, and moderate agreement for ECTb for both observers. In inter-observer repeatability, CCC revealed substantial agreement for 4DM and QPS, and poor agreement for ECTb. Regarding TDE, CCC showed substantial intra-observer repeatability for both operators using all packages, while the inter-observer repeatability was substantial for 4DM and QPS, and moderate for ECTb.In SSSext's intra-observer repeatability for 4DM, ECTb and QPS, the B-A analysis provided (mean±1.96SD of paired measurements) 0.0±4.3, 0.2±7.8, -0.6±7.6 for the experienced physician and 0.2±5.9, 0.0±7.5, -0.5±5.4 for the trainee, respectively; in inter-observer repeatability it provided 0.2±5.4, 0.1±9.6, 0.2±8.1, respectively. Regarding TDE, the B-A values for intra-observer repeatability were 0.1±5.2, 0.1±7.9, 0.1±2.8 for the experienced reader and 0.3±6.6, -0.1±6.4, -0.1±2.4 for the trainee, respectively; in inter-observer agreement the B-A provided 0.6±6.4, -0.2±10.3, -0.1±4.3, respectively. Considerable differences in intra- and inter- observer agreement were noted for the quantitative determination of defect size using widely employed software packages, suggesting limitations in the clinical use of these measurements. Quantitative perfusion SPECT appears preferable, but with no significant advantage over 4DM. There were no significant differences between the observers.