DNA sequence analysis of the trnl-trnF spacer region and real-time scorpion polymerase chain reaction were exploited for their applications in the differentiation and quantification of the traditional Chinese medicinal plants Drynaria fortunei from five related adulterants: D. mollis, D. quercifolia, D. rigidula, D. sparsisora, and Pseudodrynaria coronans. The data demonstrated that variations in the trnl-trnF spacer regions were very low at the intraspecific level but extremely high at the interspecific level, meaning that they could be easily distinguished at the DNA level. The sequence difference allowed effective and reliable differentiation of D. fortunei from adulterants by real-time scorpion PCR. Furthermore, a quantification methodology was carried out to quantify D. fortunei.