Currently, Avian Hepatitis E virus (aHEV) has emerged as a significant concern within the poultry industry, leading to elevated mortality rates characterized by enlarged livers and spleens in affected flocks. The pathological progression involves hepatic lipidosis, exudate release into the coelomic cavity, and eventual liver fracture accompanied by hemorrhages. Despite its impact, there remains a notable dearth of comprehensive information on the pathobiology and epidemiology of the virus and associated disease. This study focused on aHEV infections in commercial layers, its seroprevalence, virus isolation, designing a new diagnostic tool for detection of viral RNA, and the identification of circulating genotypes. Utilizing a commercial ELISA test, we observed approximately 30% seropositivity among all collected serum samples. In addition, we detected a positive correlation between aHEV titers and the age of the birds. Regarding viral load distribution, gallbladders exhibited the highest loads compared to the livers and spleens. The genotyping strategy was able to detect genotype 2 viruses, and for the first time in the U.S. identified genotype 3, which has significantly altered the previously established epidemiological understanding of this virus in the country. Despite efforts to isolate the virus in SPF eggs, our results were not successful. In conclusion, all these findings highlight the importance of continuing aHEV research to strengthen control strategies in the poultry sector.