Rock fractures provide dominant fluid flow pathways in the Earth's subsurface and can play an integral role in many subsurface engineering applications, such as the storage of anthropogenic carbon emissions or energy. Injection of an additional fluid into an already saturated fracture, for example, CO2 displacing brine, induces multiphase flow. The (in)stability of immiscible fluid-fluid interface interactions can exert key influences over storage/leakage capacity. Central to improving numerical and theoretical modelling of two-phase flow on the reservoir scale is obtaining a more robust understanding of drainage (displacement of a wetting by a non-wetting phase) in a single rough fracture. This requires characterisation of the pore-scale physics, which directly impacts saturation and relative permeability. We utilised state of the art 4D X-ray tomography to image drainage (2.75 μm voxel size, 1 s scan time) in a natural geological fracture at a controlled fluid flux analogous to that anticipated during CO2 fracture leakage to probe the two-phase flow response. This work can directly inform single-fracture scale simulations and elucidate larger scale (fracture-network/reservoir) two-phase flow constitutive relationships, such as capillary pressure-saturation and relative permeability.