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The introduction of glyphosate-resistant (GR) crops revolutionized weed management; however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an <i>Amaranthus hybridus</i> population (GRH)&#8212;a superweed collected in a GR-soybean field from Cordoba, Argentina&#8212;as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more ¹⁴C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA¹⁰², GTC¹⁰³ and TCA¹⁰⁶ instead of ACA¹⁰², GCG¹⁰³, and CCA¹⁰⁶, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 &#197; (wild type) to 2.93 &#197; (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH <i>A. hybridus</i> population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance.