The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha(-1) of glyphosate (i.e. Roundup) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 microM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F(1) hybrids between GR and GS horseweed. Thirty GSxGR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GSxGFP/GR F(1) hybrids produced F(2) seeds, and F(2) plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype.