Background: TIDratio indirectly reflects myocardial ischemia and is correlated with cardiacprognosis. We aimed at comparing the influence of three different softwarepackages for the assessment of TID using Rb-82 cardiac PET/CT. Methods: Intotal, data of 30 patients were used based on normal myocardial perfusion(SSS<3 and SRS<3) and stress myocardial blood flow 2mL/min/g)assessed by Rb-82 cardiac PET/CT. After reconstruction using 2D OSEM (2Iterations, 28 subsets), 3-D filtering (Butterworth, order=10, ωc=0.5), data were automatically processed, and then manually processed fordefining identical basal and apical limits on both stress and rest images.TIDratio were determined with Myometrix®, ECToolbox® and QGS®software packages. Comparisons used ANOVA, Student t-tests and Lin concordancetest (ρc). Results: All of the 90 processings were successfullyperformed. TID ratio were not statistically different between software packageswhen data were processed automatically (P=0.2) or manually (P=0.17). There was a slight, butsignificant relative overestimation of TID with automatic processing incomparison to manual processing using ECToolbox® (1.07 ± 0.13 vs 1.0± 0.13, P=0.001)and Myometrix® (1.07 ± 0.15 vs 1.01 ± 0.11, P=0.003) but not using QGS®(1.02 ±0.12 vs 1.05 ± 0.11, P=0.16). The best concordance was achieved between ECToolbox®and Myometrix® manual (ρc=0.67) processing.Conclusion: Using automatic or manual mode TID estimation was not significantlyinfluenced by software type. Using Myometrix® or ECToolbox®TID was significantly different between automatic and manual processing, butnot using QGS®. Software package should be account for when definingTID normal reference limits, as well as when used in multicenter studies. QGS®software seemed to be the most operator-independent software package, whileECToolbox® and Myometrix® produced the closest results.