The mouse continues to serve as a key model organism to study genetic and molecular features associated with human diseases. In mice and human alike, glycoproteins are prevalent and complex biomolecules central to most biological processes and tissue function. However, it remains unexplored how the mouse glycoproteome differs across tissues and genders, precluding a better understanding of murine, and, by extension, human glycobiology. To this end, we here developed a mouse tissue glycoproteome atlas (mTGA), by applying a multi-omics workflow comprising glycomics, glycoproteomics and transcriptomics approaches to comprehensively map the N-glycoproteome and the underpinning N-glycosylation machinery across 17 different tissues and plasma from adult female (n = 5) and male (n = 5) C57BL/6 mice. Collectively, a total of >26,800 unique N-glycopeptides covering >1,500 glycoproteins and an additional 5,300 different proteins groups without glycosylation were identified and quantified across all analysed samples. Excitingly, the multi-omics data revealed prominent tissue- and gender-specific glycoproteome differences across multiple molecular layers, revealing new insights into the complexity, regulation and function of glycoproteins in biological processes associated with health and disease. Notably, glycoproteomics and sialic acid speciation analysis pointed to Neu5Ac and Neu5Gc as key features driving tissue- and gender-specific glycosylation differences in salivary gland, liver, stomach, thymus, urinary bladder and plasma. Interestingly, down regulation of CMAH enzyme, responsible for Neu5Ac-to-Neu5Gc conversion, was associated with the reduced Neu5Gc levels decorating glycoproteins expressed in liver tissue and plasma from females compared to those from males. The mTGA provides a comprehensive and open-access resource of high-content glycoproteome profiling data and sialic acid signatures across mouse tissues and genders thereby serving as a valuable molecular reference map to address research questions in fundamental and applied glycobiology.