The karyotypes of three rat species from Hainan Island, China, were examined. Niviventer fulvescens (Gray, 1847) had 2n=46 and FN=64, similar to the karyotypes reported for N. fulvescens from Southeast Asia, while Niviventer lotipes (Allen, 1926) had 2n=52 and FN=66, which is distinct from the known karyotypes of other Niviventer species. Niviventer lotipes was recently considered conspecific with N. tenaster (Thomas, 1916), but the two were found to have extremely different karyotypes (2n=52 and FN=66 in N. lotipes; 2n=46 and FN=54 in N. tenaster). Therefore, in this paper N. lotipes is considered a valid species for the first time; it is distinct from N. tenaster and endemic to Hainan Island, where N. lotipes is differentiated from N. fulvescens by larger body and skull sizes, a shorter tail, and darker coloration. Rattus nitidus (Hodgson, 1845) from Hainan Island had 2n=42 and FN=62, which is similar to the reported karyotypes of conspecific populations in Southeast Asia.