Abstract BACKGROUND: In a large cropping area of northern Argentina, Sorghum halepense (Johnsongrass) has evolved towards glyphosate resistance. This study aimed to determine the molecular and biochemical basis conferring glyphosate resistance in this species. Experiments were conducted to assess target EPSPS gene sequences and 14 C‐glyphosate leaf absorption and translocation to meristematic tissues. RESULTS: Individuals of all resistant (R) accessions exhibited significantly less glyphosate translocation to root (11% versus 29%) and stem (9% versus 26%) meristems when compared with susceptible (S) plants. A notably higher proportion of the applied glyphosate remained in the treated leaves of R plants (63%) than in the treated leaves of S plants (27%). In addition, individuals of S. halepense accession R 2 consistently showed lower glyphosate absorption rates in both adaxial (10–20%) and abaxial (20–25%) leaf surfaces compared with S plants. No glyphosate resistance endowing mutations in the EPSPS gene at Pro‐101–106 residues were found in any of the evaluated R accessions. CONCLUSION: The results of the present investigation indicate that reduced glyphosate translocation to meristems is the primary mechanism endowing glyphosate resistance in S. halepense from cropping fields in Argentina. To a lesser extent, reduced glyphosate leaf uptake has also been shown to be involved in glyphosate‐resistant S. halepense . Copyright © 2011 Society of Chemical Industry