BACKGROUND: While some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks. RESULTS: Thirteen different conserved miRNAs were found to be dys-regulated in GM samples. The insecticide Bt GM variety had the most distinct miRNome. These miRNAs target a range of endogenous transcripts, such as transcription factors and nucleic acid binding domains, which play key molecular functions in basic genetic regulation. In addition, we have identified 20 potential novel miRNAs with target transcripts involved in lipid metabolism in maize. isomiRs were also found in 96 conserved miRNAs sequences, as well as potential transgenic miRNA sequences, which both can be a source of potential off-target effects in the plant genome. We have also provided information on technical limitations and when to carry on additional in vivo experimental testing. CONCLUSIONS: These findings do not reveal hazards per se but show that robust and reproducible miRNA profiling technique can strengthen the assessment of risk by detecting any new intended and unintended dsRNA molecules, regardless of the outcome, at any stage of GMO development.