Empoasca onukii (Matsuda) was analyzed and its propose to define succession rule of population and clarify the genetic differentiation and genetic diversity of different geographic populations in southestwest (Guizhou Province) of China. 516 bp segments of 16S ribosomal RNA (rRNA) gene of 120 individual E. onukii from 12 different geographic populations in Guizhou Province were tested, the genetic diversity, level of gene flow and molecular were analyzed by using software DnaSP 5.10, Arlequin3.5.1.2., 30 variable sites and 22 haplotypes were found, and Hap2 was sharing model among 12 geographic populations; haplotypes diversity and nucleotide diversity of total population was 0.5060.056 and 0.002110.00058, respectively. Genetic polymorphism was low; the level of genetic differentiation within populations (Gst=0.05227, Fst=0.04183) was low and gene flow was high. The result of Tajima's D test of all populations was minus and significant, which indicated that a lot of alleles loci in low frequency existed in total population and the phenomenon of negative selection and hitchhiking effects happened in the historical period, while the result of most different geographic populations was minus and not significant. This suggested that evolution of E. onukii in Guizhou Province was in accordance with the neutral model, the number of which was quite stable and had no population expansion, low genetic diversity among different geographic populations because the gene had exchanges frequently. The result of molecular variance (AMOVA) indicated that the genetic differentiation of E. onukii was mainly from within populations (95.82%), while not obviously among populations. Among different geographic populations, there was a certain level of genetic differentiation, and without correlation between genetic distance, gene flow and geographical distance.