Background Composite invasive and non-invasive data consistently demonstrate that resting myocardial blood flow (rMBF) in regions of known transmural myocardial scar (TMS) converge on a value of ~ 0.30 mL/min/g or lower. This value has been confirmed using the 3 most common myocardial perfusion agents ( 13 N, 15 O-H 2 O and 82 Rb) incorporating various kinetic models on older 2D positron emission tomography (PET) systems. Thus, rMBF in regions of TMS can serve as a reference “truth” to evaluate low-end accuracy of various PET systems and software packages (SWPs). Using 82 Rb on a contemporary 3D-PET-CT system, we sought to determine whether currently available SWP can accurately and precisely measure rMBF in regions of known TMS. Results Median rMBF (in mL/min/g) and COV in regions of TMS were 0.71 IQR 0.52–1.02 and 0.16 with 4DM; 0.41 0.34–0.54 and 0.10 with 4DM-FVD; 0.66 0.51–0.85 and 0.11 with Cedars; 0.51 0.43–0.61 and 0.08 with Emory-Votaw; 0.37 0.30–0.42, 0.07 with Emory-Ottawa, and 0.26 0.23–0.32, COV 0.07 with HeartSee. Conclusions SWPs varied widely in low end accuracy based on measurement of rMBF in regions of known TMS. 3D PET using 82 Rb and HeartSee software accurately (0.26 mL/min/g, consistent with established values) and precisely (COV = 0.07) quantified rMBF in regions of TMS. The Emory-Ottawa software yielded the next-best accuracy (0.37 mL/min/g), though rMBF was higher than established gold-standard values in ~ 5% of the resting scans. 4DM, 4DM-FDV, Cedars and Emory-Votaw SWP consistently resulted values higher than the established gold standard (0.71, 0.41, 0.66, 0.51 mL/min/g, respectively), with higher interscan variability (0.16, 0.11, 0.11, and 0.09, respectively). Trial registration : clinicaltrial.gov, NCT05286593, Registered December 28, 2021, https://clinicaltrials.gov/ct2/show/NCT05286593 .