Abstract An Echinochloa colona population from Western Australia has evolved resistance to glyphosate. This current study investigates the physiological, molecular and biochemical basis of glyphosate resistance in this population. To minimise genetic differences the susceptible (S) and resistance (R) phenotypes were isolated from within this resistant population. The S phenotype was found to accumulate significantly more shikimate in the leaf tissue than the R phenotype following glyphosate treatment. Target-site EPSPS gene sequencing revealed no resistance mutations and EPSPS gene expression was similar between the S and R phenotypes. Thus, glyphosate resistance in this population is unlikely target-site based. Similarly, there were no significant differences between the S and R phenotypes in glyphosate leaf uptake and translocation at the whole plant level. It is also unlikely that the resistance is associated with glyphosate metabolism as no major glyphosate metabolites were detected in leaf tissue of the S and R phenotypes. Despite much effort the exact glyphosate resistance mechanisms in this R population remain unclear, and novel resistance mechanisms are to be determined.