Invariant chain (Ii) isoform, through its thyroglobulin-like (Tg) domain, inhibits cysteine proteases during antigen presentation in vertebrates. In birds, the Ii of Muscovy Duck (MDIi) has 2 forms: MDIi-1 and MDIi-2 (MDIi isoform). To understand the genetic information and expression characteristics of MDIi-2, polymerase chain reaction, and bioinformatic analysis were performed for MDIi-2 from healthy adult Muscovy Duck. The full-length MDIi-2 cDNA sequence was found to be 1377-base pairs, encoding a 285-amino acid protein. MDIi-2 contains 63 amino acids with an insertion sequence in the Tg domain. MDIi-2 shares high identity (72.51-94.74%) with the same protein in other birds. The Tg domain of MDIi-2 is highly conserved and showed relatively high identity (96.83%) among all tested birds. The molecular structure of the Tg domain supports this conservation. MDIi-2 expression was measured in various tissues using real-time quantitative polymerase chain reaction. Similar to MDIi-1, MDIi-2 was detected in all tissues but at different levels.