Background <p>The development of genetically modified crops has made significant contributions to addressing challenges in disease and pest control as well as weed management. However, the integration of exogenous DNA into plant genomes may introduce unintended effects. In this study, we investigated three transgenic papaya lines approved for biosafety certification in China—Huanong No. 1, YK1601, and 55-1—through analyses at both DNA and transcriptional levels.</p> Results <p>Examination of T-DNA insertion sites and sequences revealed that integration events not only caused local genomic deletions but also incorporated unintended fragments derived from the transformation vector or host genome. Moreover, T-DNA integration displayed site preference, favoring AT-rich genomic regions. Compared with the 55-1 line generated via particle bombardment, the Huanong No. 1 and YK1601 lines produced through <i>Agrobacterium</i>-mediated transformation exhibited relatively minor structural alterations at insertion sites. Transcriptomic analysis further indicated that T-DNA integration did not significantly affect the expression of flanking genes near the insertion loci. Nevertheless, several disease-resistance genes associated with plant immunity and defense responses were upregulated in the transgenic lines.</p> Conclusions <p>Collectively, this study provides a systematic molecular characterization of T-DNA integration in multiple transgenic papaya lines and evaluates the broader transcriptomic impacts. These findings not only provide a molecular theoretical basis for the biosafety assessment of transgenic papaya but also offer valuable references for evaluating unintended effects in other genetically modified crops.</p>