In cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis, it is critical to choose a suitable pair of restriction enzymes for tagging sites in cDNA for amplification. Possibility of production of chimeric fragments from cDNA-AFLP analysis remains to be researched. The objectives of this study were to detect an efficient restriction enzyme combination for cDNA-AFLP analysis when Festuca species was used as template, and to evaluate the identity of transcript-derived fragments (TDFs) from cDNA-AFLP analysis. We found that NspI coupled TaqI was a pair of highly efficient enzymes by generating a much higher number of TDFs than the commonly used EcoRI and TaqI. This was the first study to apply NspI for AFLP analysis, prompting that this enzyme may have valuable application potential for other species. The identity of TDF was evaluated by sequencing a TDF and comparing it with the sequence of the template cDNA. The result showed that the chimeric fragments derived from ligation between digested fragments was generated and could not be eliminated by increasing adapter concentration. Although the existence of chimeric fragments should be carefully considered, the unexpected sequence in the chimeric TDF may not seriously influence the sequencing and BLAST searching analyses.