Transgene escape risks are evaluated by assessing the frequency of transgenic hybrids within populations sexually compatible with those genetically modified. We examined the pollen-mediated transgene flow among lettuce varieties in Italy. Transgenic ‘Luxor’ lines harboured the arabidopsis KNAT1 gene, which caused leaf trait alterations, and the bacterial NPTII gene for resistance to kanamycin. Donor homozygous lines (KNAT1:NPTII/KNAT1:NPTII) were used in two trials: in glasshouse, ‘Luxor’ transgenic and conventional plants were spaced at 15 cm; in field, a transgenic ‘Luxor’ donor island was set up and surrounded by 10 pollen receiver varieties sited at 0.5, 11 and 22 m. Progenies of recipient cultivars were screened by kanamycin assays, leaf alterations, transgene transcription and integrity. The out-crossing rate (OCR) was calculated as the frequency of KNAT1:NPTII/− and δKNAT1:NPTII/− genotypes because KNAT1 impair (indicated with δ) was observed to vary from the 0.03% to 0.36%. Indoor, ‘Luxor’ intra-varietal OCR was 0.41 ± 0.11%; outdoor, the average inter-varietal OCR was 0.49%, 0.071% and 0.035% for plants at 0.5, 11 and 22 m, respectively.