Gene copy number proliferation is one mechanism amongst several involved in the evolution and spread of resistance. Pinning down the relative impact of gene copy number proliferation and generalized stress response pathways remains elusive due to phenotype complexity and modulation of selection pressure over spatio-temporal scales. Furthermore, specific genomic process(es) underlying rapid adaptive evolution during resistance, particularly copy number multiplication-mediated resistance at the population level, has been difficult to capture. To understand the genomic origins of gene copy number proliferation during increased resistance amplitudes and the subsequent population level processes expanding resistant biotypes, we investigated genus Amaranthus and populations of the species with the most rapidly spreading resistance, Amaranthus palmeri. The primary mechanism underlying glyphosate resistance in A. palmeri is massive duplication and random spread of EPSPS gene copy numbers across the genome. EPSPS produces 5-enolpyruvylshikimate-3-phosphate synthase, an essential enzyme in the aromatic amino acid biosynthesis pathway. Outside of glyphosate-resistant biotypes of some species, EPSPS is present in single copy, is highly conserved, and contains a binding domain that is the target site of glyphosate across genus Amaranthus, in ancestral A. palmeri, and other plant families. In this study, we estimated EPSPS gene copy numbers, EPSP synthase abundances, glyphosate resistance bioassays, and genetic population structures of A. palmeri across the southeastern U.S.A. We then compared the divergence population genetics profiles of effective population sizes, divergence times, and the rates and patterns of gene flow of EPSPS versus randomized non-target genes. Our results indicate independent origins of glyphosate resistance in A. palmeri and that recently-founded resistant populations have differing levels of variance in massive amplification of the EPSPS gene (from ancestral single copy state to upwards of 400 copies). This multi-population study of A. palmeri with differing levels of glyphosate resistance and genetic structures indicates gene amplification as a dominant source of resistance in some populations in contrast to other, more generalized stress response processes in other populations. Taken together, there seems to be alternative routes of glyphosate resistance in A. palmeri which may, in part, be shaped or modulated by population structure.