Rodents, globally overpopulated, are important source for zoonotic disease transmission to humans, including Enterocytozoon bieneusi (one of the most prevalent zoonotic pathogen). Here, we studied the prevalence and performed genetic analysis of E. bieneusi in rodents from the Hainan province of China by the amplification of the internal transcribed spacer (ITS) region of rDNA of E. bieneusi using PCR. Six hundred and three fresh fecal samples were gathered from 369 wild rats, 117 bamboo rats, 93 Asiatic brush-tailed porcupine and 24 red-bellied squirrels. The average rate of infection of E. bieneusi was 15.8% (95/603) with 18.7% (69/369) in wild rats, 11.9% (25/210) in farmed rodents and 4.2% (1/24) for red-bellied squirrels. Sixteen E. bieneusi genotypes were identified, including nine known genotypes (D, Type IV, PigEBITS7, Peru8, Peru11, ESH02, S7, EbpA and CHG5), and seven novel genotypes (HNR-I to HNR-VII). Genotype D (44.2%, 42/95) predominated, followed by PigEBITS7 (20.0%, 19/95), HNR-VII (15.8%, 15/95), Type IV (5.3%, 5/95), HNR-III (2.1%, 2/95), HNR-VI (2.1%, 2/95) and each of the remaining 10 genotypes (1.1%, 1/95). This is the first report on the identification of E. bieneusi in rodents from Hainan, China. The zoonotic potential of the identified E. bieneusi genotypes suggested that the rodents posed a serious threat to the local inhabitants. Thus, measures need to be taken to control the population of wild rats in the areas investigated in this study, along with identification of safe methods of disposal of farmed rodent feces. Additionally, the local people should be made aware of the risk of disease transmission from rodents to humans.