The possible transfer and accumulation of novel DNA and/or proteins in food for human consumption derived from animals receiving genetically modified (GM) feed is at present the object of scientific dispute. A number of studies failed to identify GM DNA in milk, meat, or eggs derived from livestock receiving GM feed ingredients. The present study was performed in order to: (i) develop a valid protocol by PCR and multicomponent analysis for the detection of specific DNA sequences in milk, focused on GM maize and GM soybean; (ii) assess the stability of transgenic DNA after pasteurization treatment and (iii) determine the presence of GM DNA sequences in milk samples collected from the Italian market. Results from the screening of 60 samples of 12 different milk brands demonstrated the presence of GM maize sequences in 15 (25%) and of GM soybean sequences in 7 samples (11.7%). Our screening methodology shows a very high sensitivity and the use of an automatic identification of the amplified products increases its specificity and reliability. Moreover, we demonstrated that the pasteurization process is not able to degrade the DNA sequences in spiked milk samples. The detection of GM DNA in milk can be interpreted as an indicator of fecal or airborne contamination, respectively, with feed DNA or feed particles, although an alternative source of contamination, possibly recognizable in the natural environment can be suggested. Further studies, performed on a larger number of milk samples, are needed to understand the likely source of contamination of milk collected from the Italian market.