Although the Mallard (Anas platyrhynchos) is considered an important maintenance host for low pathogenic avian influenza (LPAI) viruses, viral cell tropism and pathology in naturally infected birds are largely unknown. In August 2006, we collected 19 free-living hatch-year Mallards that were positive for LPAI virus by real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) in combined oropharyngeal and cloacal swabs. We investigated virus infection and associated lesions in the digestive and respiratory tracts by RRT-PCR, virus culture, immunohistochemistry (IHC), and histology. By RRT-PCR, 15 birds were positive in cloacal bursa, colon/cloaca, or both, and three were positive in lungs. Virus was isolated from eight birds and typed as H2N3 (three birds), H3N3 (two birds), H3N8 (one bird), H4N6 (one bird), and H?N3 (one bird). By IHC, birds were positive in the cloacal bursa (eight birds), colon (three), cecum (two), or ileum (one). Cell types infected were superficial epithelial cells of the bursa and epithelial cells of the intestinal villi and, less commonly, mucosal glands. By histology, there was no evidence of lesions associated with LPAI virus infection. These results show that epithelia of the cloacal bursa and of the lower intestine are important sites of natural LPAI virus infection in free-living hatch-year Mallards. The lack of lesions associated with this infection suggests that there is a strong selection by LPAI virus to cause minimal virulence in this maintenance host species.