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In recent decades, cereal agriculture across main producing areas in Spain has progressively adopted direct sowing, associated with an increased use of herbicides in pre-sowing. Weedy species from genus <i>Avena</i> have been observed after the application of glyphosate in wheat. Here, <i>Avena fatua</i> (two accessions), <i>Avena byzantina</i> and <i>Avena sterilis</i> subsp. <i>sterilis</i>, one accession each, were taxonomically characterized by a biometric study. Dose-response trials confirmed that one <i>A. fatua</i> accession evolved to resistance, because it was four times more resistant (R) than the others, ascribed as susceptible (S). In addition, based on LD₅₀, <i>A. byzantina</i> and <i>A. sterilis</i> had low susceptibility to glyphosate, with 604 and 458 g ae ha⁻¹. Shikimic acid accumulation was able to discriminate between high susceptible (<i>A. fatua</i> (S)), low susceptible (<i>A. byzantina</i> and <i>A. sterilis</i>), and low resistant (<i>A. fatua</i> (R)) populations. On the other hand, the study revealed that <i>A. fatua</i> (R), <i>A. byzantina</i> and <i>A. sterilis</i> had low foliar uptake and decreased movement of glyphosate. In addition, the metabolism study showed less metabolite accumulation in <i>A. byzantina</i> and <i>A. fatua</i> (S). However, at 96 h after glyphosate application, <i>A. fatua</i> (R) and <i>A. sterilis</i> were able to convert more than 30% of glyphosate to aminomethylphosphonic acid (AMPA) and sarcosine. Enzyme basal activity and I₅₀ values predicted high affinity between the herbicide and the target enzyme for all accessions, thus indicating that a target-site (TS) mechanism is probably not involved in the differences in glyphosate susceptibility. In closing, non-target site (NTS) mechanisms could participate both in <i>A. fatua</i> R to glyphosate, and low susceptibility in <i>A. byzantina</i> and <i>A. sterilis</i> from Spain.