Amaranthus palmeri is the most prominent invasive weed in agricultural land from North America, partly due to its propensity to evolve resistance to multiple herbicide sites of action. In the last two decades, reports of this species have increased throughout the American continent and occasionally in other continents. In 2007, A. palmeri populations were found in three localities in northeastern Spain, and they are still present today. To determine whether these three populations resulted from a common or independent introduction events—and when and from where they could have occurred—research was carried out aiming to characterize the resistance profile and mechanisms to 5-enolpyruvylshikimate-3-phosphate synthase-and acetolactate synthase (ALS)-inhibiting herbicides and to analyze the relationship between these three populations using inter simple sequence repeat DNA fingerprinting. Dose–response trials confirmed that the three populations were susceptible to glyphosate but resistant to nicosulfuron-methyl. Resistance to ALS inhibitors was due to several amino acid substitutions in positions Pro197, Trp574 and Ser653. Moreover, the substitutions Ser653Ile and Pro197Thr are described for the first time in this species. At field-labeled rates, all populations were fully controlled with alternative herbicides with other sites of action. Amaranthus palmeri individuals were clustered in three groups based on unweighted pair group method with arithmetic mean analysis, which corresponded to the three sampled populations, with a 67% of genetic relationship among them. Considering this high genetic variability and the different positions and amino acid substations found between populations, it was hypothesized that different colonization events occurred from the American continent probably prior to the introduction of glyphosate resistant crops. Prevention from new introductions is warranted because new herbicide resistance traits could arrive, complicating the management of this invasive weed species, while managing or eradicating the already established populations.