Palmer amaranth is native to the United States, but was discovered in 2015 in Brazil. Palmer amaranth populations in Brazil were very difficult to control using glyphosate, which resulted in many changes to standard weed management practices. A genotyping assay was used to confirm that the population detected in Mato Grosso State, Brazil, was correctly identified as Palmer amaranth and that it was not tall waterhemp. Greenhouse dose–response curves and shikimate accumulation assays showed that the Brazilian population was highly resistant to glyphosate, with an LD50 value (3,982 g glyphosate ha−1) more than twice the typical use rates and very little shikimate accumulation at 1 mM glyphosate concentrations in a leaf-disk assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone acetolactate synthase (ALS) inhibitor herbicides. The resistance mechanisms in the Brazilian population were identified as increased EPSPS gene copy number for glyphosate resistance (between 50- and 179-fold relative EPSPS gene copy number increase) and two different alleles for target-site mutations in the ALS gene (W574L and S653N). These results confirm the introduction of Palmer amaranth to Brazil using a genetic marker for species identification, as well as resistance to glyphosate and ALS inhibitors.