Abstract T-DNA transformation is prevalent in Arabidopsis research and has expanded to a broad range of crops and model plants. While major progress has been made in optimizing the Agrobacterium-mediated transformation process for various species, a variety of pitfalls associated with the T-DNA insertion may lead to the misinterpretation of T-DNA mutant analysis. Indeed, secondary mutagenesis either on the integration site or elsewhere in the genome, together with epigenetic interactions between T-DNA inserts or frequent genomic rearrangements, can be tricky to differentiate from the effect of the knockout of the gene of interest. These are mainly the case for genomic rearrangements that become balanced in filial generations without consequential phenotypical defects, which may be confusing particularly for studies that aim to investigate fertility and gametogenesis. As a cautionary note to the plant research community studying gametogenesis, we here report an overview of the consequences of T-DNA-induced secondary mutagenesis with emphasis on the genomic imbalance on gametogenesis. Additionally, we present a simple guideline to evaluate the T-DNA-mutagenized transgenic lines to decrease the risk of faulty analysis with minimal experimental effort.