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Enterocytozoon bieneusi is an important pathogen that is responsible for over 90% of documented cases of human microsporidiosis worldwide, causing a threat to public health and husbandry development. In immunocompromised patients, it can cause persistent diarrhea, wasting diathesis and malabsorption, and developing life-threatening chronic diarrhea. However, there was little information on the prevalence and multilocus genotypes of E. bieneusi in diarrheic pigs in three provinces of southern China. In this study, 1254 fecal samples of diarrheic pigs were collected from 37 pig farms in Hunan, Jiangxi, and Fujian provinces in southern China, and were investigated the prevalence and genotypes of E. bieneusi by nested polymerase chain reaction (PCR) based on the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA gene. The overall prevalence of E. bieneusi was 5.7% (72/1254) in three provinces. Furthermore, the difference was statistically significant (P < 0.001) in the prevalence of E. bieneusi in age groups. ITS sequence analysis revealed that 13 E. bieneusi genotypes were identified, including 8 known genotypes (EbpC, n = 30; Henan-IV, n = 21; CH5, n = 6; EbpA, n = 3; KIN-1, n = 2; O, n = 1; GX3, n = 1; CHS5, n = 1) and 5 novel genotypes (JX1, JX2, JX3, FJ1 and FJ2), and the genotype EbpC was the preponderant genotype. Phylogenetic analysis indicated that all genotypes of E. bieneusi were clustered as the zoonotic group 1. Moreover, a high genetic diversity of E. bieneusi were identified in this study, which the 64, 57, 52, and 64 samples were identified by multilocus sequence typing (MLST) at MS1, MS3, MS4, and MS7 loci, respectively. Then, 45 samples were successfully amplified and sequenced at four loci, forming 41 distinct multilocus genotypes (MLGs). These findings suggest that diarrheic pigs may potentially threaten to transmit E. bieneusi to humans, revealing E. bieneusi genetic variability in diarrheic pigs in three provinces of southern China. This article is protected by copyright. All rights reserved.