Ten Holstein/Friesian cows with an average liveweight of 630 kg and producing 25.3 kg milk/day received a total mixed ration (TMR) in which the forage component formed 55% of total DM and contained non-genetically modified (GM) grass and maize silage in a 1:3 DM ratio. In study weeks 1-3 the TMR DM also contained non-GM supplements of 18.5% cracked wheat and 26.1% rapeseed meal. In weeks 4-12 soyabean meal, genetically modified (CP4EPSPS) to be herbicide tolerant, replaced rapeseed meal at 26.1% of the total diet in weeks 4-5 and 13.9% of the total diet in weeks 6-12. Weekly milk samples were taken from all cows. Samples were spiked with DNA extracted from the soyabean meal to establish the limit of detection (LOD) of transgenic DNA using a polymerase chain reaction (PCR) analyses designed to detect transgenic DNA fragments smaller than 200 bp. Subsequent PCR analyses carried out in duplicate on TMR and milk samples collected at weeks 3, 4 and 12 of the study used three DNA primer sets to establish the presence or absence of transgenic DNA. The LOD of transgenic DNS in milk was 7.5 μg/l. Feed and milk samples analysed at week 3 were negative. The TMR feed samples at weeks 4 and 12 were positive for CP4EPSPS soyabean meal DNA, but all milk samples were negative. The results showed that transgenic DNA could not be detected in milk from cows receiving upto 26.1% of their diet DM as herbicide (glyphosate)-tolerant soyabean meal.