Rat hepatitis E virus (rat HEV, Rocahepevirus genotype C1) represents a potential zoonotic threat, but its epidemiological and evolutionary characteristics in small mammals remain poorly understood, especially in regions with complex geography. Between 2022 and 2024, we collected 818 small mammals from seven border counties and cities in Yunnan, China. Rat HEV RNA was detected by RT-PCR, risk factors were assessed using binary logistic regression, and full genomes were sequenced for phylogenetic and molecular clock analysis. The overall prevalence of rat HEV was 6.23% (51/818), with significantly higher odds observed in Gengma and Heqing counties, in oriental house rat (Rattus tanezumi) and Chevrieri’s field mouse (Apodemus chevrieri), in residential habitats, and at mid-high altitudes (all p < 0.001). The 51 partial genomic sequences (RdRp gene) obtained in this study clustered within Rocahepevirus, forming two distinct subclades associated with host species. The two complete genomes, GS188 and GS197 from Rattus tanezumi, were classified as subtypes C1b and C1d, respectively. Bayesian analysis estimated that GS197 diverged from a closely related Rattus tanezumi-derived strain around 1998, while GS188 diverged from a lineage containing shrew and human strains around 1931. These findings reveal a relatively high prevalence and substantial genetic diversity of Rochepevirus in southwestern Yunnan, suggesting human-influenced transmission dynamics and a potential for cross-species infection.

Rat hepatitis E virus (rat HEV, Rocahepevirus genotype C1) represents a potential zoonotic threat, but its epidemiological and evolutionary characteristics in small mammals remain poorly understood, especially in regions with complex geography. Between 2022 to 2024, we collected 818 small mammals from seven border counties and cities in Yunnan, China. Rat HEV RNA was detected by RT-PCR, risk factors were assessed using binary logistic regression, and full genomes were sequenced for phylogenetic and molecular clock analysis. The overall prevalence of rat HEV was 6.23% (51/818), with significantly higher odds observed in Gengma and Heqing counties, in oriental house rat (Rattus tanezumi) and Chevrieri's field mouse (Apodemus chevrieri), in residential habitats, and at mid-high altitudes (all P < 0.001). The 51 genomic sequences obtained clustered within HEV-C1, forming two distinct subclades associated with host species. Two complete genomes (GS188 and GS197) were classified as subtypes C1b and C1d, closely related to strains detected in shrews from Guangdong and in human cases from Hong Kong, with divergence times estimated around 1931 and 1998, respectively. These findings reveal a relatively high prevalence and substantial genetic diversity of HEV-C1 in southwestern Yunnan, suggesting human-influenced transmission dynamics and a potential for cross-species infection.