A truncated ORF2 capsid HEV antigen derived from a genotype 3 strain was developed in insect cells and insect larvae, and compared with the Sar55 antigen and a commercial ELISA. The antigen expressed in insect cells showed a better correlation with Sar55 (kappa value (k)=0.84) than the insect larvae antigen (k=0.69), and a better reproducibility as indicated by the intra and interplate variation coefficients. Commercial ELISA designed for human diagnosis but adapted to animal use using specific secondary antibodies demonstrated to have a very low sensitivity. The insect cell expressed antigen was used to develop an ELISA to detect anti-HEV-IgG in serum samples of different domestic animal and rodents. Seropositivity in the studied animal populations was 71.4% for pigs, 0.60% for goats, 1.92% for sheep, and 11.11% for cats. None of the 1170 cattle samples or 166 rodent samples analyzed was positive.