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A study about the influence of polyaxial (true-triaxial) stresses on the permeability of a three-dimensional (3D) fractured rock layer is presented. The 3D fracture system is constructed by extruding a two-dimensional (2D) outcrop pattern of a limestone bed that exhibits a ladder structure consisting of a “through-going” joint set abutted by later-stage short fractures. Geomechanical behaviour of the 3D fractured rock in response to in-situ stresses is modelled by the finite-discrete element method, which can capture the deformation of matrix blocks, variation of stress fields, reactivation of pre-existing rough fractures and propagation of new cracks. A series of numerical simulations is designed to load the fractured rock using various polyaxial in-situ stresses and the stress-dependent flow properties are further calculated. The fractured layer tends to exhibit stronger flow localisation and higher equivalent permeability as the far-field stress ratio is increased and the stress field is rotated such that fractures are preferentially oriented for shearing. The shear dilation of pre-existing fractures has dominant effects on flow localisation in the system, while the propagation of new fractures has minor impacts. The role of the overburden stress suggests that the conventional 2D analysis that neglects the effect of the out-of-plane stress (perpendicular to the bedding interface) may provide indicative approximations but not fully capture the polyaxial stress-dependent fracture network behaviour. The results of this study have important implications for understanding the heterogeneous flow of geological fluids (e.g. groundwater, petroleum) in subsurface and upscaling permeability for large-scale assessments., Hydrogeology Journal, 25 (8), ISSN:1431-2174, ISSN:1435-0157