Ostrinia furnacalis(Guenée), the Asian corn borer, is one of the most important agricultural pests in China. It can damage crops during different crop growth stages, and can affect the yield and quality of maize. In addition, the pest has a wide geographic distribution and host range. With global warming and the expansion of corn acreage in recent years, the occurrence of the pest and associated damages have been increasing. Studying the genetic structure of the Asian corn borer is necessary for understanding the pest's evolution and migration in Shandong, a critical corn production region in China. In order to reveal the genetic diversity and structure of O. furnacalis in Shandong, China, and throughout the world, we analyzed 340 mtCOII sequences of O. furnacalis obtained both from the present study and GenBank (updated to October 1^st, 2011). Of the mtCOII sequences analyzed, 214 sequences were obtained from GenBank while 126 sequences were obtained during the present study. The mtCOII sequences were aligned using MEGA5.05 and were then checked for indels and numts. Using DnaSP 5.0, a set of genetic parameters for mtCOII were estimated including: the number of polymorphic (segregating) sites (S); the total number of mutations (η); the average number of nucleotide differences (K); the number of haplotypes (H); the haplotype diversity (Hd);the nucleotide diversity (π), defined as the average number of pairwise nucleotide differences per site; and the nucleotide diversity with Jukes and Cantor correction for different host collections from Shandong Province. The results revealed that there were a total of 62 haplotypes, among which the haplotype H17 was the most widely distributed. Using the 62 haplotypes, a phylogenetic tree was constructed with the maximum likehood (ML) method; two clades (Clade Ⅰ and Clade Ⅱ) were revealed. The populations from Shandong Province consisted of two clades, although Clade I was the dominant clade. The haplotypes from Shandong were distributed randomly among 17 populations with no obvious geographical pattern. The haplotype diversity (Hd) of the populations from Shandong ranged from 0.333 to 0.889, and the Hd of the entire populations in Shandong was 0.695. The nucleotide diversity (π) of the populations from Shandong ranged from 0.00061 to 0.00809 and the π of total populations was 0.00424. Analysis of molecular variance (AMOVA) showed that 79.42% of the total genetic variance was contributed by the inter-clade variation and only 20.58% contributed by the intra-clade variation. Finally, to rapidly differentiate the two clades, a PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) method was developed. The PCR-RFLP method of differentiating the two clades within O. furnacalis will be helpful for future research on the biological and ecological differences between the two clades. Our work revealed that the incidence of Clade Ⅱ was less than Clade Ⅰ. This might suggest that they are different in biology, ecology and physiology. Such differences could affect the clade's geographic distributions and population diffusions. These studies' results serve as a guide for the sustainable control of the pest, however these issues need to be further studied.