Background Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g. insecticidal and/or herbicide tolerance). Moreover, data on expression levels for GM events are usually required for approval, but these are rarely disclosed or they are considered insuﬃcient. On the other hand, biosafety regulators do not consider epigenetic regulation (e.g. DNA methylation, ncRNAs and histone modiﬁcations), which are broadly known to aﬀect gene expression, within their risk assessment analyses. Here we report the results of a DNA methylation (bisulﬁte sequencing) and transgene transcript accumulation (RT‑qPCR) analysis of four Bt‑expressing single transgenic maize hybrids, under diﬀerent genetic backgrounds, and a stacked transgenic hybrid expressing both insecticidal and herbicide tolerance traits. Results The results showed diﬀerences in cytosine methylation levels in the FMV promoter and cry2Ab2transgene of the four Bt‑expressing hybrid varieties. The comparison between single and stacked hybrids under the same genetic background showed diﬀerences in the 35S promoter sequence. The results of transgene transcript accumulation levels showed diﬀerences in both cry1A.105 and cry2Ab2transgenes among the four Bt‑expressing hybrid varieties. The comparison between single and stacked hybrids showed diﬀerence for the cry2Ab2 transgene only. Conclusions The results show diﬀerences in DNA methylation patterns in all varieties, as well as in transgene transcript accumulation levels. Although the detection of changes in DNA methylation and transgenic accumulation levels does not present a safety issue per se, it demonstrates the need for additional studies that focus on detecting possible safety implications of such changes.