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Objective： To demonstrate the polymorphism of α chain of bovine major histocompatibility complex (BoLA) class Ⅰmolecule and domain binding constant chain (Ii). Methods： Total 75 BoLA Iα genes were obtained from three Huaibei cattle and analyzed by molecular biology software； the genes of typical BoLA Iα domains and Ii were cloned, and then inserted into prokaryotic expression plasmid. After induced protein expression； the domains of BoLA Ⅰα chain binding to Ii were detected by pull-down method and Western blot. The recombinant eukaryotic expression plasmids were constructed and the co-localization of BoLA Iα segments with Ii was observed by laser confocal microscopy. Results： Firstly, it was found that there were at least 5 kinds of BoLA Iα in the cloned gene sequence, which were highly polymorphic and they were mainly distributed in the antigen peptide binding region (PBR) of BoLA Ⅰ (α1α2) and cytoplasmic region. Secondly, the prokaryotic recombinant plasmids containing BoLA Ⅰα1α2α3, BoLA Ⅰα1α2 or BoLA Ⅰ α3 were constructed, then they were respectively induced to express and purified, in which, the BoLA Ⅰ α1α2α3 and BoLA Ⅰα1α2 had the activity of binding to Ii. Finally, in 293T cells BoLA Ⅰα1α2α3 or BoLA Iα1α2 was found that could co-localize with Ii, while a single BoLA Iα3 could not. Conclusion： BoLA Ⅰα gene is highly polymorphic. BoLA Ⅰα1α2 is a functional fragment that binds to Ii and co-locates intracellular. [ABSTRACT FROM AUTHOR]