Previous studies have documented the occurrence of glyphosate-resistant(GR) goosegrass(Eleusine indica(L.) Gaertn.) and, in at least some cases, resistance is due to an altered target site. Research was performed to determine if an altered target site was responsible for GR in a Tennessee, United States goosegrass population(Tenn GR). DNA sequencing revealed a mutation in Tenn GR plants conferring the Pro106 Ser 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) substitution previously identified in other GR populations. F_2 populations were derived from Tenn GR plants crossed with plants from a glyphosate-susceptible population(Tenn GS) and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F_2 populations segregated 1:2:1 sensitive:intermediate:resistant in response to a selective dose of glyphosate, and these responses co-segregated with the EPSPS genotypes(PP106, PS106, and SS106). To separately investigate the effect of the Pro106 Ser substitution on GR, glyphosate dose-response curves and 50% effective dose(ED_(50)) values were compared among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Pro106Ser EPSPS mutation is solely responsible for GR in the TennGR goosegrass population. Previous studies have documented the occurrence of glyphosate-resistant (GR) goosegrass (Eleusine indica (L.) Gaertn.) And, in at least some cases, resistance was due to an altered target site. site was responsible for GR in a Tennessee, United States goosegrass population (Tenn GR). DNA sequencing revealed mutation in Tenn GR plants conferring the Pro106 Ser 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) substitution previously identified in other GR populations. F_2 populations were derived from Tenn GR plants crossed with plants from a glyphosate-susceptible population (Tenn GS) and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F2 population segregated 1: 2: 1 sensitive: intermediate: resistant in response to a selective dose of glyphosate, and these responses co-segregated with the EPSPS genotypes (PP106, PS106, and SS106). The spread on GR, glyphosate dose-response curves and 50% effective dose (ED_ (50)) values ​​were among among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Pro106Ser EPSPS mutation is not solely responsible for GR in the TennGR goosegrass population.