To enrich gene polymorphism ofDuMHCI and provide data for further studies on disease resistance, 14DuMHCI genes from Weishan Ma duck and Cherry Valley duck were cloned, and their characterization were investigated. The overall conservation of the 14 alleles could be observed within the sequences, and relative conservation were also displayed in the peptide-binding domain and CD8 interaction sites. Based on full-length amino acid homology, MHC class I from different duck lines could be divided into 13 gene groups and three novel gene groups existed.Moreover, 14 key variable residues corresponding to gene groups division were exhibited on the homology modelling constructed based on the resolved protein structure of DuMHC I. This study explicit the characteristics of DuMHC I in the two duck lines and could contribute to design effective diagnostics and vaccines for the species against various infections.