The physiological and molecular bases of glyphosate resistance in one susceptible (S) and four resistant (R) Conyza bonariensis biotypes (sampled in orchards from Andalusia, Spain) were investigated. Resistance index (RI) values of the four R biotypes ranged between 2.9 and 5.6. The main physiological difference between S and R biotypes was the dissimilar mobility of glyphosate in the whole plant. In R biotypes, the herbicide was translocated less from leaves to culm and root, and more from culm to leaves compared with the S biotype. The upward mobility of glyphosate via xylem suggests that the herbicide may be sequestered to the apoplast or the vacuole. The hypothesis of an insensitive 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was provisionally discarded on the basis of shikimate accumulation in R plant tissues after glyphosate treatment. At the molecular level, the relative abundance of EPSPS mRNA prior to glyphosate treatment was approximately double in two R biotypes compared with the S standard and the other R biotypes. Moreover, the two R biotypes having both no translocation and doubled EPSPS mRNA levels had also the highest RI. These results suggest that two factors may be related to glyphosate resistance in the R biotypes: (i) impaired translocation and (ii) high basal EPSPS transcript levels. The comparison between these findings and earlier results on glyphosate resistance mechanism in Conyza canadensis biotypes from the USA, suggests that similar agronomic factors (repeated application of glyphosate, no crop and herbicide rotation, no tillage) have selected similar traits on different genetic pools of the resistance-prone Conyza genus.