Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to the formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and renders their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered the investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to characterize a previously unmapped MUC3B gene located at the q22 locus on chromosome 7, within a cluster of four structurally related membrane mucin genes that we name the MUC3 cluster. We found that MUC3B shares high sequence identity with the known MUC3A gene and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12, and MUC17 in the human MUC3 cluster are expressed in intestinal epithelial cells (IECs). Our results complete existing genetic gaps in the MUC3 cluster which is a conserved genetic unit in vertebrates. We anticipate our results to be the starting point for the detection of disease-associated polymorphisms in the human MUC3 cluster. Moreover, our study provides the basis for the exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.