Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization, which can enrich the complexity of transcriptomes and proteomes. In this study, the first exonization event was detected when the modified rice EPSPS marker gene was inserted with the Ac transposon 5' end, which provided a splice donor site to yield abundant novel transcripts. To assess the contribution of splice donor and acceptor sites of transposon sequences, we inserted a Ds element into each intron of the EPSPS marker gene. This process yielded 14 constructs, with the Ds transposon inserted in the forward and reverse direction in each of the 7 introns of the EPSPS marker gene. The constructs were transformed into tobacco plants, and novel transcripts were identified by RT-PCR with specific primers. Exonization of Ds in EPSPS was biased towards providing splice donor sites of the inserted Ds sequence. Additionally, when the Ds inserted in reverse direction, a continuous splice donor consensus region was determined by offering 4 donor sites in the same intron. Information on these exonization events may help enhance gene divergence and functional genomic studies.