This study describes an efficient approach to the development of DNA markers for use in distinguishing the
Scrophularia species that have been used as useful medicinal crops. In order to distinguish Scrophularia species, DNA
sequences of rpl-5 region in mitochondrial DNA of Scrophularia species were analysed for detecting sequence variations, and
the PCR-RFLP method was applied for developing practicable DNA marker patterns. Several DNA variations were
detected by the sequence comparison of rpl-5 region among Scrophularia species. Genetic relationship analysis of Scrophularia
species was carried out based on these DNA variations. DNA variations of rpl-5 region were revealed that it was significantly
efficient in genetic relationship analysis of Scrophularia species. In addition, Scrophularia species tested in this study
were completely discriminated by four polymorphic genotypes by PCR-RFLP combined with Tsp509 I (^AATT) restriction
enzyme. Our results suggested that DNA sequence variations of rpl-5 region were sufficiently useful for genetic relationship
analysis of Scrophularia species. Polymorphic genotypes by PCR-RFLP using the Tsp509 I enzyme will be useful for discrimination
of Scrophularia species as a practicable DNA markers.