Abstract Purpose Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatic disease worldwide with functional impairment of the mitochondria occurring from early stages. Technetium-99m methoxy-isobutyl-isonitrile (99mTc-MIBI) is a lipophilic agent trapped in the mitochondria. This study aims to evaluate the utility of 99mTc-MIBI heart/liver uptake ratio in screening for NAFLD during myocardial perfusion imaging (MPI). Methods 70eligible patients underwent a two-day rest/stress 99mTc-MIBI scan with a 2-minute planar image acquired in rest phase, at 30, 60 and 120 minutes post radiotracer administration. Heart/liver uptake ratio was calculated by placing identical regions of interest on the heart and liver dome. All patients underwent liver ultrasound and were allocated into groups A, having NAFLD, and B, healthy individuals without NAFLD. Results Mean count per pixel heart/liver ratios gradually increased over time in either groups; nonetheless the values were significantly higher in group A, regardless of acquisition timing; with the P-value equal to 0.007, 0.014 and 0.010 at 30, 60 and 120 minutes, respectively. Conclusions Determining 99mTc-MIBI heart/liver uptake ratio during rest phase in patients undergoing MPI may be a useful, non-invasive screening method for NAFLD; with no additional cost, radiation burden or adverse effects in these patients.