Recently, several incidents of glyphosate failure on junglerice [Echinochloa colona (L.) Link] have been reported in the midsouthern United States, specifically in Mississippi and Tennessee. Research was conducted to measure the magnitude of glyphosate resistance and to determine the mechanism(s) of resistance to glyphosate in E. colona populations from Mississippi and Tennessee. ED50 (dose required to reduce plant growth by 50%) values for a resistant MSGR4 biotype, a resistant TNGR population, and a known susceptible MSGS population were 0.8, 1.62, and 0.23 kg ae ha−1 of glyphosate, respectively. The resistance index calculated from the these ED50 values indicated that the MSGR4 biotype and TNGR population were 4- and 7-fold, respectively, resistant to glyphosate relative to the MSGS population. The absorption patterns of [14C]glyphosate in the TNGR and MSGS populations were similar. However, the MSGS population translocated 13% more [14C]glyphosate out of the treated leaf compared with the TNGR population at 48 h after treatment. EPSPS gene sequence analyses of TNGR E. colona indicated no evidence of any point mutations, but several resistant biotypes, including MSGR4, possessed a single-nucleotide substitution of T for C at codon 106 position, resulting in a proline-to-serine substitution (CCA to TCA). Results from quantitative polymerase chain reaction analyses suggested that there was no amplification of the EPSPS gene in the resistant populations and biotypes. Thus, the mechanism of resistance in the MSGR population (and associated biotypes) is, in part, due to a target-site mutation at the 106 loci of the EPSPS gene, while reduced translocation of glyphosate was found to confer glyphosate resistance in the TNGR population.