Nanocrystalline lithium peroxide (Li2O2) is considered to play a critical role in the redox chemistry during the discharge–charge cycling of the Li–O2 batteries. In this report, a spatially resolved, real-time synchrotron X-ray diffraction technique was applied to study the cyclic formation/decomposition of Li2O2 crystallites in an operating Li–O2 cell. The evaluation of Li2O2 grain size, concentration, and spatial distribution inside the cathode is demonstrated under the actual cycling conditions. The study not only unambiguously proved the reversibility of the Li2O2 redox reaction during reduction and evolution of O2, but also allowed for the concentration and dimension growths of the peroxide nanocrystallites to be accurately measured at different regions within the cathode. The results provide important insights for future investigation on mass and charge transport properties in Li2O2 and improvement in cathode structure and material design.