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BACKGROUND: Gene amplification has been shown to provide resistance to glyphosate in several weed species, including Hordeum glaucum populations in South Australia. The stability of 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene copies in resistant populations in the presence or absence of glyphosate selection has not been determined. RESULTS: Applying glyphosate to a cloned plant resulted in an increase in resistance and EPSPS copy number in the progeny of that plant compared to the untreated clone. The LD50 (herbicide concentration required for 50% mortality) increased by 75% to 79% in the progeny of the treated clones compared to the untreated in both populations (YP‐17 and YP‐16). EPSPS copy number estimates were higher in treated individuals compared to untreated individuals with an average of seven copies compared to six in YP‐16 and 11 compared to six in YP‐17. There was a positive correlation (R2 = 0.78) between EPSPS copy number and LD50 of all populations. CONCLUSION: EPSPS gene copy number and resistance to glyphosate increased in H. glaucum populations under glyphosate selection, suggesting the number of EPSPS gene copies present is dependent on glyphosate selection. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]