In this study the modified wall degradation hypotonic method was used for unlocking the chromosome number and the karyotype of Urophysa rockii Ulbr., a plant species of which is distributed in an extremely restricted area. The 5S rDNA sites on chromosomes were also analyzed of Urophysa rockii Ulbr. via fluorescence in situ hybridization (FISH). The results indicated that Urophysa rockii Ulbr. is a diploid species with 14 chromosomes (n = 7). The relative length of chromosomes was 2n=14=2L+2M2+10M1, and no satellite chromosome was detected with the karyotype formula. The average centromere index and karyotype asymmetry coefficient were 44.09 and 55.91, respectively. The karyotype belonged to 1A type, suggesting that phylogenetic evolution is relatively primitive. Furthermore, two 5S rDNA sites were detected close to the centromere of the long arm of chromosome 1. Taken together, these results may provide shed-light on systematology and cytology of Urophysa rockii Ulbr.