Islands are often regarded as natural laboratories for the study of biological evolution by ecologists and evolutionary biologists. Based on microsatellite molecular marker technology, we analyzed the genetic diversity of three Rattus losea populations in Zhoushan Archipelago, and calculated the related genetic parameters and the coefficient of genetic differentiation with the software POPGEN 32 and ARLEQUIN 3.0, respectively. We also calculated the minimum paternal value with the software GERUD 2.0, and found the existence of multiple paternity. The results were as following: (1) The average observed number of alleles (NA) and the average effective number of alleles (Ne) were 8.792 and 5.494, respectively (Table 2); the average expected heterozygosity (He) and the average observed heterozygosity (Ho) were 0.821 and 0.956, respectively; the polymorphism information content (PIC) were larger than 0.5 (Table 2), which indicated that three populations had a high degree of genetic diversity. (2) The average genetic differentiation value among three populations was 0.040, and the value between Taohua island and Cishan island was the largest (Fst = 0.052), showing a moderate level of differentiation. The percentage of variation among three populations accounted for 3.98% (Table 4), which indicated that the differentiation occurred mainly within the populations. (3) Six out of the fourteen pregnant rats (43%) had their embryos with multiple paternity (Fig. 3). The minimum paternal value was 2 (Table 5), and the embryos of the other pregnant rats had only one father. This study proposed the existence of multiple paternity phenomenon of R. losea populations in Zhoushan Archipelago, and provided useful information for the study of reproductive strategy and pest control of R. losea populations.