The objective of this study was to evaluate the carry-over of transgenic soyabean and maize DNA in samples of milk deriving from different groups of cows fed with either genetically modified (GM) or GM-free feed. Also, to understand the possible source of such contamination of milk which can be of endogenous or exogenous origin (contamination from GM feed containing ‘dust or aerosols’). The milk and feed samples were taken during routine practices of the dairy farms in order to be as close as possible to real condition. In total 66 samples of cow’s milk and 120 samples of feed (GM, GM-free and organic feed) were collected in six Italian farms with different farming systems (organic and conventional) and types of barn stalls (milking area contiguous or separated from feeding station). The quantitative Real-Time PCR analysis of samples confirmed the presence of GM soya and maize in GM labeled feed and their absence in organic/GM-free feed. In the latter group, neither transgenic nor endogenous soyabean DNA was detected in the milk samples as expected. The limit of detection was estimated by spiking whole milk samples with GM plant DNA before DNA extraction. The smallest concentration of soyabean DNA required for detection was 1 ng · ml^–1 of milk for lectin gene which corresponded to about 900 copies per ml of milk. No milk samples of GM-fed cows was found suspicious for the presence of recombinant DNA within the limit of detection. This means that neither transfer of genetic material nor aerosol contamination from feed to milk can be shown in the investigated husbandry system.