In parallel to the increased utilization of cross-sectional imaging, the incidence of small renal masses has steadily risen in recent years. At the present time, anatomical imaging techniques are limited in their ability to differentiate benign from malignant renal masses. Moreover, renal mass biopsy has a high non-diagnostic rate, poor negative-predictive ability, and carries potential risks as well as substantial costs. As a result, benign renal masses are often unnecessarily resected for the false presumption of cancer. 99mTc-sestamibi single photon emission computed tomography/computed tomography (SPECT/CT) is a molecular imaging test that allows for the differentiation of benign renal oncocytomas and hybrid oncocytomic/chromophobe tumours apart from malignant renal cell carcinomas on the basis of differences in mitochondrial content as well as multidrug resistant pump expression. Herein, we review the principles of 99mTc-sestamibi SPECT/CT administration and image interpretation for the purpose of renal mass characterization.