Hepatitis E virus (HEV) infects several animal species that act as zoonotic reservoirs for viral transmission. Solid and liquid residues from infected animals could lead to HEV contamination of food and surface waters. Evidence of human HEV infection through ingestion of seafood (shellfish, mussels) has been reported. Dolphins generally feed on fish and squid but are able to adapt to an environment and consume whatever prey is available. Clinical signs of infected dolphins include lethargy, inappetence, behavioral aberrations and increased serum alanine aminotransferase (ALT). The dolphins examined in this study were maintained at the National Aquarium, Havana, Cuba. A total of 31 dolphins were evaluated for HEV markers. Sera were collected and screened for total immunoglobin (Ig) anti-HEV. Sera and liver homogenate were tested for HEV RNA by nested RT-PCR using primers targeting the open reading frame 1. Phylogenetic analysis was performed using partial nucleotide sequences at the amplified RNA-dependent RNA polymerase gene. Total anti-HEV Ig was detected in 32.2% (10 of 31), and 16.1% (5 of 31) of these dolphins were positive by both serology and HEV RNA testing. Nucleotide sequence analyses revealed that HEV strains identified in dolphins were genotype 3. This virus may represent an environmental contamination of food or wastewater as a source of HEV exposure and infection. Our findings provide evidence that HEV is associated with liver disorders in cetaceans and that it is advisable to screen for exposure of this virus in captive dolphins, particularly animals with elevated serum ALT or compromised liver function test results of undetermined cause.