Management of agroecosystems with herbicides imposes strong selection pressures on weedy plants leading to the evolution of resistance against those herbicides. Resistance to glyphosate in populations of Lolium perenne L. ssp. multiflorum is increasingly common in California, USA, causing economic losses and the loss of effective management tools. To gain insights into the recent evolution of glyphosate resistance in L. perenne in perennial cropping systems of northwest California and to inform management, we investigated the frequency of glyphosate resistance and the genetic diversity and structure of 14 populations. The sampled populations contained frequencies of resistant plants ranging from 10% to 89%. Analyses of neutral genetic variation using microsatellite markers indicated very high genetic diversity within all populations regardless of resistance frequency. Genetic variation was distributed predominantly among individuals within populations rather than among populations or sampled counties, as would be expected for a wide-ranging outcrossing weed species. Bayesian clustering analysis provided evidence of population structuring with extensive admixture between two genetic clusters or gene pools. High genetic diversity and admixture, and low differentiation between populations, strongly suggests the potential for spread of resistance through gene flow and the need for management that limits seed and pollen dispersal in L. perenne.