The objective of this study was to determine patterns of avian HEV infection in naturally infected chicken farms. A total of 310 serum samples and 62 pooled fecal samples were collected from 62 chicken flocks on seven commercial in-line egg farms in the Midwestern United States and tested for avian HEV circulation. Serum samples were tested for presence of anti-avian HEV IgY antibodies by a fluorescent microbead immunoassay (FMIA) which was developed for this study. The FMIA was validated using archived samples of chickens with known exposure (n = 96) and compared to the result obtained with an enzyme-linked immunosorbent assay (ELISA) based on the same capture antigen. There was an overall substantial agreement between the two assays (κ = 0.63) with earlier detection of positive chickens by the FMIA (P = 0.04). On the seven farms investigated, the overall prevalence of anti-avian HEV IgY antibodies in serum samples from commercial chickens was 44.8% (20-82% per farm). Fecal samples were tested for avian HEV RNA by a nested reverse-transcriptase PCR. The overall detection rate of avian HEV RNA in fecal samples was 62.9% (0-100% per farm). Sequencing analyses of partial helicase and capsid genes showed that different avian HEV genotype 2 strains were circulating within a farm. However, no correlation was found between avian HEV RNA detection and egg production, egg weight or mortality. In conclusion, avian HEV infection is widespread among clinically healthy laying hens in the United States.