Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. The increasing incidence of non-O157 STEC has posed a great risk to public health. Besides the Shiga toxin (Stx), the adherence factor, intimin, coded by eae gene plays a critical role in STEC pathogenesis. In this study, we investigated the prevalence and polymorphisms of eae gene in non-O157 STEC strains isolated from different sources in China. Among 735 non-O157 STEC strains, eae was present in 70 (9.5%) strains. Eighteen different eae genotypes were identified in 62 eae-positive STEC strains with the nucleotide identities ranging from 86.01% to 99.97%. Among which, seven genotypes were newly identified in this study. The eighteen eae genotypes can be categorized into five eae subtypes, namely β1, γ1, ε1, ζ3 and θ. Associations between eae subtypes/genotypes and serotypes as well as origins of strains were observed in this study. Strains belonging to serotypes O26:H11, O103:H2, O111:H8 are associated with particular eae subtypes, i.e., β1, ε1, θ, respectively. Most strains from diarrheal patients (7/9, 77.8%) carried eae-β1 subtype, while most isolates from cattle (23/26, 88.5%) carried eae-ζ3 subtype. This study demonstrated a genetic diversity of eae gene in non-O157 STEC strains from different sources in China.