In this work, we systematically compared O-acetylated sialoglycopeptides (O-AcSGPs) among sera of human, rat, and mouse to reveal differences and diversities of sialic acid types and O-acetylation patterns in different species. We utilized our high-resolution glycoproteomic approaches to identify numerous O-acetylated N-glycans in rat and mouse sera, and described their structures and distributions across different glycoproteins.