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Glyphosate-resistant giant ragweed (Ambrosia trifida L.) was first identified in Canada in 2008. Although early studies attributed resistance in this species solely to non-target site mechanisms, the presence of a proline (P) to serine (S) mutation at position 106 of EPSPS2 in common and giant ragweed has recently been reported. The objective of this research was (i) to determine whether a P106S mutation is present in historical samples of giant ragweed seed collected from the site of the first report of glyphosate resistance, and (ii) to determine the frequency and distribution of P106S in resistant and susceptible biotypes collected as part of historical surveys throughout southwestern Ontario. [ABSTRACT FROM AUTHOR]