This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS–AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive β-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Lea) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.