Dermatophagoides farinae (Acari: Pyroglyphidae) has been regarded as an important source of indoor allergens due to its prevalence and close proximity to humans. This study serves as an introductory probe into the population genetic structure of D. farinae, for which a few related studies of population genetic structure are available. In this study, the genetic structure of seven D. farinae populations in China was investigated based on the 372 bp mitochondrial cytochrome b (cytb) gene. A total of 29 haplotypes of the 68 cytb gene sequences were detected. The network diagram could be divided into Branch I (regions north of the Yangtze River: SJZ, CZ, FY and HN) and Branch II (regions south of the Yangtze River: AQ, WX and WH). Analysis of molecular variance (AMOVA) conducted according to the results of the network diagram revealed major genetic differentiation among groups, indicating that a significant genetic structure existed among groups. The results of population genetic structure could be largely attributed to geographical isolation. The existence of gene flow in a few populations may be influenced by grain trade activities. This study may facilitate a better understanding of the population genetic structure of D. farinae and could be utilized as the foundation for the development of efficient management strategies for D. farinae.