Abstract Background Myocardial perfusion imaging by positron emission tomography (PET-MPI) is the current gold standard for quantification of myocardial blood flow. ¹⁸F-flurpiridaz was recently introduced as a valid alternative to currently used PET-MPI probes. Nonetheless, optimum scan duration and time interval for image analysis are currently unknown. Further, it is unclear whether rest/stress PET-MPI with ¹⁸F-flurpiridaz is feasible in mice. Methods Rest/stress PET-MPI was performed with ¹⁸F-flurpiridaz (0.6-3.0 MBq) in 27 mice aged 7–8 months. Regadenoson (0.1 µg/g) was used for induction of vasodilator stress. Kinetic modeling was performed using a metabolite-corrected arterial input function. Image-derived myocardial ¹⁸F-flurpiridaz uptake was assessed for different time intervals by placing a volume of interest in the left ventricular myocardium. Results Tracer kinetics were best described by a two-tissue compartment model. K₁ ranged from 6.7 to 20.0 mL·cm⁻³·min⁻¹, while myocardial volumes of distribution (V_T) were between 34.6 and 83.6 mL·cm⁻³. Of note, myocardial ¹⁸F-flurpiridaz uptake (%ID/g) was significantly correlated with K₁ at rest and following pharmacological vasodilation for all time intervals assessed. However, while Spearman’s coefficients (r_s) ranged between 0.478 and 0.681, R² values were generally low. In contrast, an excellent correlation of myocardial ¹⁸F-flurpiridaz uptake with V_T was obtained, particularly when employing the averaged myocardial uptake from 20 to 40 min post tracer injection (R² ≥ 0.98). Notably, K₁ and V_T were similarly sensitive to pharmacological vasodilation induction. Further, mean stress-to-rest ratios of K₁, V_T, and %ID/g ¹⁸F-flurpiridaz were virtually identical, suggesting that %ID/g ¹⁸F-flurpiridaz can be used to estimate coronary flow reserve (CFR) in mice. Conclusion Our findings suggest that a simplified assessment of relative myocardial perfusion and CFR, based on image-derived tracer uptake, is feasible with ¹⁸F-flurpiridaz in mice, enabling high-throughput mechanistic CFR studies in rodents.