In this study we demonstrate the feasibility of following up a chemical reaction by single crystal x-ray (synchrotron) diffraction under operando conditions, carried out in a specially designed electrochemical cell mounted on the BM01A at the European Synchrotron Radiation Facility (ESRF). We investigated in detail the electrochemical oxidation of SrFeO 2.5 to SrFeO 3 on a spherical single crystal of 70 µ m diameter by in situ diffraction at an ambient temperature. Complete data sets were obtained by scanning the whole reciprocal space using a 2M Pilatus detector, resulting in 3600 frames with a resolution of 0.1° per data set, each obtained in 18 min. The crystal was mounted in a specially designed electrochemical cell with 1N KOH used as the electrolyte. During the electrochemical oxidation, the reaction proceeds following the phase sequence SrFeO 2.5 /SrFeO 2.75 /SrFeO 2.875 /SrFeO 3 , structurally accompanied by establishing a complex series of long-range oxygen vacancy ordering, which gets instantly organized at ambient temperature. The topotactic reaction pathway is discussed in terms of the evolution of the twin domain structure. The formation of SrFeO 2.875 is accompanied by the formation of diffuse streaks along the [1 0 0]-direction of the perovskite cell, reaching high d -spacings. The diffuse streaks are discussed and are thought to originate from a modified twin structure induced by the SrFeO 2.75 to SrFeO 2.875 transition, and the associated changes in the domain structure, developed during the oxygen intercalation. We equally analysed and discussed in detail the twin structure of all the title compounds. We confirm the ground state of SrFeO 2.5 is able to adopt the Imma space group symmetry, showing stacking faults of the tetrahedral layers along the stacking axis of the brownmillerite unit cell, indicated by the 1D diffuse rods. We showed that in situ single crystal diffraction has huge potential in the study of non-stoichiometric compounds under operando conditions, in order to obtain structural information i.e. about diffuse scattering, and microstructural information related to domain effects such as twinning—information far beyond that which powder diffraction methods allow us to obtain