A failure of the EPSPS-inhibiting herbicide glyphosate to control several populations of Hordeum murinum subsp. leporinum (or H. murinum) occurred in southern Spain after more than fifteen applications in both crop (olive, orchards, and citrus) and non-crop (dry areas, roadsides and ditches) areas. Eight out of 18 populations studied were resistant (R) to glyphosate with R factors higher than four based on GR50. These populations also had the highest values of LD50 and the lowest levels of shikimic acid accumulation. Two adjuvants tested increased glyphosate efficacy in both susceptible (S) and R populations thanks to better spray foliar retention. Moreover, PS I-, PS II-, and ACCase-inhibiting herbicides, in pre- or post-emergence, proved to be the best chemical alternatives with different sites of action (SoA) to control both S and glyphosate-R populations. This study represents the first report worldwide of glyphosate resistance in H. murinum found in very different crop and non-crop areas from southern Spain. To design chemical strategies to implement integrated weed management programs for glyphosate-R H. murinum, both adjuvants and herbicides with alternative SoA as well as application timings should be considered.