Single crystals of Ca4Fe2Mn0.5Ti0.5O9 have been synthesized using a flux method. The structural characterization using single-crystal X-ray diffraction revealed the space group Amma and unit-cell dimensions of a = 5.3510 (6), b = 26.669 (3), c = 5.4914 (6) Å. The structure is isotypic with Sr3NdFe3O9 [Barrier et al. (2005). Chem. Mater. 17, 6619–6623] and exhibits separated brownmillerite-type layers. One-dimensional diffuse scattering shows that the unit cell is doubled along c by alternating the intra-layer order of tetrahedral chains, causing stacking faults along the b direction. A computer simulation was performed, proving that the observed intensity variations along the diffuse scattering rods originates from two different local structures depending on the configuration of the tetrahedral chains. Selected-area electron diffraction experiments exhibit well ordered regions characterized by satellite reflections corresponding to two different superstructures. Both superstructures can be described using the superspace group A21/m(0βγ)0s, with γ = 0.5 and β ≃ 0.27 or β = 0.