The purpose of this study was to characterize the T-DNAs introduced into the transgenic OsCK rice, as part of a biosafety evaluation. Choline Kinase (CK) gene is upregulated in the transgenic OsCK rice. We identified the insertion sites, flanking sequences, structures and sequences of the inserted T-DNAs. Based on the adaptor-ligation PCR, we found that the right border of the T-DNA was inserted at position no. 129971, on BAC clone OSJNBa0014J14 of chromosome 10. The flanking sequences of the left border region of the T-DNA (which was later identified as a region harboring a 1-kb long deleted sequence), could not be identified by various PCR-based trials. However, it was finally identified with whole-genome shotgun sequencing, using an Illumina sequencer. The result indicated that one of the T-DNAs was inserted into the CaMV 35S promoter region, whereas the other T-DNA was introduced at position 128947 on OSJNBa0014J14 clone, with an inverse orientation. During the insertion process, a 1024-bp-long chromosome sequences flanked by the right border of the T-DNA region was deleted. A 370-bp long left border region and 199-bp long right border region corresponding to the matrix attachment region (MAR) sequences of the T-DNA were also deleted. Collectively, these results indicate that whole-genome shotgun sequencing is a useful tool to reveal the detailed sequences and structures of the introduced T-DNAs, especially in the case of multiple T-DNA insertions. The expenses incurred on genome sequencing can be easily compensated by minimizing the time and efforts invested in conventional molecular analyses.