Adaptive evolution occurs rapidly under the environment demand and can be an important element in the evolution of animals. Former researches about the adaptive evolution of animals are most focus on the changes of morphological traits, but few studies have compared the behavior. Relative to morphological traits, we know little about the adaptive evolution of complex behavior in nature. To understood the difference for adaptive evolution, we tested the nesting behavior in two subspecies of Microtus fortis, M. f. fortis from Qingtongxia of Ningxia Hui Autonomous Region and M. f. calamorum that from Dongting Lake of Hunan province. In the present study, two methods were used to evaluate the nesting behavior of each vole: the 48 h nesting test for the ability of using nesting materials, which materials were contained in the cage, and we grade a point of the nest that build after 48 hours; the 144 h test for the ability of collecting nesting materials, which the weight of materials used by rodents can be recorded every 24 hours and during the test. Differences of nesting points and nesting materials weight between the two subspecies were analyzed by One-way ANOVA test. We found that two subspecies of M. fortis are both behaved strong capacity of nesting, actively acquired nesting materials, and build good quality nests. However, the nesting behavior is different significantly between two subspecies of M. fortis in their nesting point (P < 0.01, Fig. 2) and the nesting material weight (P < 0.05, Fig. 3). There is a significant difference in nesting point (P < 0.05) and nesting material weight (P < 0.05) between females and males of M. f. fortis; but the differences of the nesting behavior between the male and female individuals did not appear in M. f. calamorum. Our results indicated both the two subspecies of M. fortis have strong nesting requirements, and M. f. fortis own better nesting ability in terms of nesting materials usage and collection rather than M. f. calamorum. For these two closely related species, the difference of nesting behavior can also reflect the adaptive evolution to different environments.