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Mucus layers (McLs) are on the front line of the human defense system that protect us from foreign abiotic/biotic particles (e.g., airborne virus SARS-CoV-2) and lubricates our organs. Recently, the impact of McLs on human health (e.g., nutrient absorption and drug delivery) and diseases (e.g., infections and cancers) has been studied extensively, yet their mechanisms are still not fully understood due to their high variety among organs and individuals. We characterize these variances as the heterogeneity of McLs, which lies in the thickness, composition, and physiology, making the systematic research on the roles of McLs in human health and diseases very challenging. To advance mucosal organoids and develop effective drug delivery systems, a comprehensive understanding of McLs' heterogeneity and how it impacts mucus physiology is urgently needed. When the role of airway mucus in the penetration and transmission of coronavirus (CoV) is considered, this understanding may also enable a better explanation and prediction of the CoV's behavior. Hence, in this Review, we summarize the variances of McLs among organs, health conditions, and experimental settings as well as recent advances in experimental measurements, data analysis, and model development for simulations.