Objective To analyze the genetic diversity and differentiation of different geographic populations of Tyrophagus putrescentiae. Methods From June to July 2018, mite specimens were collected from flour and rice mills in Wuhu City (WH) and Fuyang City (FY) in Anhui Province, Shijiazhuang City (SJZ) and Zhangjiakou City (ZJK) in Hebei Province, to screen for copra mites identified by morphology and cytochrome C oxidase subunit 1 (cox1) gene sequence. Mitochondrial cytochrome b (Cytb) and ribosomal DNA internal transcriptional spacer (ITS) of the copra mites were amplified by PCR and sequenced. Using Chromas 2 and DNAStar 1.00 software were used to proofread and concatenate gene sequences. DnaSP 5.10.00 software to calculate haplotype diversity (Hd) and nucleotide polymorphism (Pi) of various mite populations, MEGA 10.2 software package to analyze population genetic variation and differentiation index (F_st) and gene flow (N_m), Arlequin 3.1 software to calculate Tajima’s D value, neutral tests to estimate Fu’s FS value, and analysis of molecular variance to assess genetic variation. To construct haplotype network diagram, Network 10.2 program was used based on the median-joining method. Haplotype phylogenetic tree was constructed using maximum likelihood method (ML). Results The T. putrescentiae mite was elliptical in shape, with soft skin, milky white or yellow-brown colour, and highly variable or degraded mouthparts. The consistency between the cox1 sequence obtained in this study and the cox1 sequence in GenBank (login number: LC190838.1) was greater than 98%. The length of the Cytb gene in the sample of T. putrescentiae was 372 bp. Among the 16 haplotypes (H1-H16), only H4 was a shared haplotype (shared by 9 individuals from the WH and FY populations), while the rest were exclusive haplotypes. The Hd of the four geographical populations was relatively high, with an overall value of 0.895 (> 0.5), with the WH population having the highest Hd (0.867) and the SJZ population having the lowest Hd (0.464). Based on Cytb sequence analysis, it was found that the genetic diversity of the four geographical populations of T. putrescentiae was relatively high (Pi > 0.005). Molecular analysis of variance showed that the F_st of the four geographical populations of T. putrescentiae was > 0.15 (P < 0.05). The neutral test results showed that the Tajima’s D value and the Fu’s FS value were -0.737 22 and 2.336 33, respectively (both P > 0.05). The haplotype network diagram was consistent with the results of the phylogenetic tree, where individuals from the four geographic populations were interwoven and distributed, with only a few individuals from the ZJK population clustered into another branch. The length of the ITS gene sequence was 1259-1405 bp, and all 32 haplotypes (G1-G2) were exclusive haplotypes. The Hd of the four geographical populations was relatively high, with an overall value of 1.000 (> 0.5) compared to the respective values. Based on ITS sequence analysis, it was also found that the genetic diversity of the four geographical populations of T. putrescentiae was high (Pi > 0.005), with F_st > 0.25 (P < 0.05). The Tajima’s D value and Fu’s FS value were 2.030 29 and 3.044 54, respectively (both P > 0.05). The haplotype network diagram was consistent with the results of the phylogenetic tree. The haplotypes of WH, FY and SJZ populations were clustered into one branch, while the haplotypes of ZJK were clustered into a single branch. Conclusion 4 geographic populations of T. putrescentiae are highly polymorphic, showing significant genetic differentiation. FY population might expanded to WH population historically, and there have been high level of partial gene exchange between different geographic populations of copra mites, but no obvious geographical distribution pattern was found.