The spatially heterogeneous distribution of biofouling in spiral wound membrane systems restricts (i) the water distribution over the membrane surface and therefore (ii) the membrane-based water treatment. The objective of the study was to assess the spatial heterogeneity of biofilm development over the membrane fouling simulator (MFS) length (inlet and outlet part) at three different cross-flow velocities (0.08, 0.12 and 0.16 m/s). The MFS contained sheets of membrane and feed spacer and simulated the first 0.20 m of spiral-wound membrane modules where biofouling accumulates the most in practice. In-situ non-destructive oxygen imaging using planar optodes was applied to determine the biofilm spatially resolved activity and heterogeneity. Comparison of the inlet and outlet position of the MFS showed a more (i) heterogeneous biofilm distribution and a (ii) higher biological activity at the inlet side (first 2.5 cm) for all cross-flow velocities. The lowest cross-flow velocity had the highest biomass activity particularly at the inlet side. A better characterization of biofilm development, including the factors that influence the biofilm spatial heterogeneity in membrane systems with time, may help to develop effective strategies for biofouling control in membrane systems. (C) 2016 Elsevier B.V. All rights reserved.