The generation of transgenic apple plants relies on the molecular analysis of transgene integration and expression based on polymerase chain reaction (PCR) analysis, blotting techniques and enzymatic assays on vitro leaves of putative transgenic regenerates. In order to assess the uniformity and the stability of transfer DNA (T-DNA) integration and gene expression, we studied 26 transgenic apple lines carrying the attacin E gene from Hyalophora cecropia, the β-glucuronidase gene, and the nptII gene. Plants were evaluated using standard molecular techniques, such as PCR, Southern blot, reverse transcription PCR (RT-PCR) and Enzyme Linked Immunosorbent Assay (ELISA), and propagated in vitro on non-selective antibiotic-free media for four years to mimic natural conditions in the field. In some T-lines transgene integration and expression did not remain stable; differences were also found between distinct plants of a single T-line. Individual plants with partially or completely silenced transgenes were identified as well as plants with non-detectable T-DNA. Several lines appeared chimeric or partially silenced. Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or cells with silenced transgenes. This should be taken into consideration, especially in the case of vegetatively propagated trees, where non-transformed or silenced plant parts could mistakenly be used as propagation material.