Niviventer confucianus sacer Thomas, 1908, which has been regarded as a subspecies of N. confucianus , was found to be a distinct species from N. confucianus based on molecular, karyotyping, and morphological characteristics in this study. Niviventer c. sacer was found to belong to a distinct phylogenetic clade in phylogenetic tree constructed using the mitochondrial gene Cytb, it clustered with N. bukit (Bonhote, 1903) from Vietnam and N. confucianus (Milne-Edwards, 1871) from Yunnan, but showed a distant relationship with N. confucianus from adjacent areas. The genetic distance between N. c. sacer and N. confucianus was more than 5.8%, reaching the level of interspecific differentiation. The species delimitation indicates that N. c. sacer is a monophyletic group. The karyotype of N. c. sacer (FN = 55, 8m+4st+32t+X(sm)Y(t)) differed from that of N. confucianus (FN = 59, 6m+4sm+2st+32t+X(sm)Y(t)). In terms of morphological features, the length of incisive foramen (LIF) and length of auditory bulla (LAB) of N. c. sacer is significantly larger than that of N. confucianus and N. bukit ( P < 0.05) and the proportion of white tail tip to total tail length is significantly longer at N. c. sacer (≥ 1/3) than that at N. confucianus (≤ 1/3). Therefore, integrated analysis confirmed that N. c. sacer is a distinct species of genus Niviventer rather than a subspecies of N. confucianus or N. bukit , namely N. sacer , which is only distributed in Shandong.