© 2015 Society of Chemical Industry. BACKGROUND: This study confirms and characterises glyphosate resistance in two polyploid Echinochloa colona populations from north-eastern Australia. RESULTS: Glyphosate dose response revealed that the two resistant populations were marginally (up to twofold) resistant to glyphosate. Resistant plants did not differ in non-target-site foliar uptake and translocation of 14C-glyphosate, but contained the known target-site 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutation Pro-106-Thr and/or Pro-106-Leu. Although plants carrying either a single or two EPSPS mutations were glyphosate resistant relative to the susceptible population, they were still controlled at the field rate of glyphosate (450 g a.e. ha-1) when treated under warm conditions (25/20 °C). However, when treated in hot conditions (35/30 °C), most mutant resistant plants (68%) can survive the field rate, and an increase (2.5-fold) in glyphosate LD50 was found for both the R and S populations. CONCLUSIONS: This study shows that one or two EPSPS Pro-106 mutations are insufficient to confer field-rate glyphosate resistance in polyploidy E. colona at mild temperatures. However, control of these mutant plants at the glyphosate field rate is poor at high temperatures, probably owing to reduced glyphosate efficacy. Therefore, glyphosate should be applied during relatively mild (warm) temperature periods in the summer growing season to improve E. colona control.