Glycosylation is an abundant and important protein modification with large influence on the properties and interactions of glycoconjugates. Human plasma N-glycosylation has been the subject of frequent investigation, revealing strong associations with physiological and pathological conditions. Less well-characterized is the plasma N-glycosylation of the mouse, the most commonly used animal model for studying human diseases, particularly with regard to differences between strains and sexes. For this reason, we used MALDI-TOF(/TOF)-MS(/MS) assisted by linkage-specific derivatization of the sialic acids to comparatively analyze the plasma N-glycosylation of both male and female mice originating from BALB/c, CD57BL/6, CD-1, and Swiss Webster strains. The combined use of this analytical method and the recently developed data processing software named MassyTools allowed the relative quantification of the N-glycan species within plasma, the distinction between α2,3- and α2,6-linked N-glycolylneuraminic acids (due to respective lactonization and ethyl esterification), the detection of sialic acid O-acetylation, as well as the characterization of branching sialylation (Neu5Gcα2,3-Hex-[Neu5Gcα2,6-]HexNAc). When analyzing the glycosylation according to mouse sex, we found that female mice present a considerably higher degree of core fucosylation (2–4-fold depending on the strain), galactosylation, α2,6-linked sialylation, and larger high-mannose type glycan species compared with their male counterparts. Male mice, on the contrary, showed on average higher α2,3-linked sialylation, branching sialylation, and putative bisection. These differences together with sialic acid acetylation proved to be strain-specific as well. Interestingly, the outbred strains CD-1 and Swiss Webster displayed considerably larger interindividual variation than inbred strains BALB/c and CD57BL/6, suggesting a strong hereditable component of the observed plasma N-glycome.