With the unique ecosystems, islands are often regarded as natural laboratories for studying biological evolution by ecologists and evolutionary biologists. In recent years, the increasing number of scientists have paid attentions to island biogeography. In this study, the genetic variation characteristics were analyzed for Rattus losea populations in 8 islands with different area and distance in Zhoushan archipelago, which is of great significance to study the population dynamics and population differentiation of island rule. Mitochondrial molecular marker technique was used to obtain the 815 bp D-loop region sequence by PCR. A total of 15 haplotypes were identified in 330 samples. The average nucleotide diversity (P_i) was 0.001 and the average haplotype diversity (H_d) was 0.364, which revealed that the genetic diversity of Rattus losea populations in Zhoushan Archipelago had a low level. The Tajima's D neutral test showed that the parameters of the populations were negative values except for XPZ population (P < 0.01), indicating that the populations were naturally selected and experienced the population expansion. AMOVA results showed that the average genetic differentiation index (74.5%) among populations was at a higher level, indicating the genetic variation mainly occurred in the populations. Phylogenetic tree and median network constructed on the basis of mitochondrial D-loop region sequences indicated that the 8 Rattus losea populations originated from two matrilinear systems. Beside, the results of mental test results showed that there was a significantly positive correlation between genetic distance and geographic distance (r=0.6077, P=0.004), and no significant correlation was found between genetic diversity and island area (r=0.6255, P=0.1840). The results can be used as a reference for the microevolution of Rattus losea populations and the evolution theory of some island species.