Abstract: Transcriptome sequencing based on high throughput has provided an opportunity for rapidly developing more molecular markers. 92042086 reads were obtained in this research based on transcriptome sequence of Arnebia szechenyi, then acquiring 332 lowcopy genes by searching 158446 unigene of Aszechenyi. In order to verify DNA polymorphism, this research randomly selected 30 lowcopy genes as designing primers and 9 individuals in three natural populations of Aszechenyi were detected by PCR amplification. It was found that 16 pairs of 30 lowcopy gene primers could amplify steadily single target band. After that, 7 fragments randomly selected from 16 primers were sequenced and used to calculate DNA polymorphism, the results showed that mean value of polymorphic sites based on 7 primers is 657, hyplotype is 514, hyplotype diversity (HD) was from 0389 to 0944, nucleotide diversity (Pi) was from 000148 to 001723. Our findings indicated that lowcopy nuclear genes developed in this study based on transcriptome sequencing have relatively high polymorphism, and it could be validly used for the study of populations genetic and phylogeography. Furthermore, these lowcopy nuclear genes could be applied to the fields of phylogeny reconstruction and speciation of Arnebia.