Thiolated chitosans constitute an integral part of designated “thiomers”, which are thiolated polymers widely investigated for non-invasive drug delivery. In brief, thiomers display thiol-group-bearing ligands on their polymer backbone. Through thiol/disulfide exchange reactions and/or a simple oxidation process, disulfide bonds are formed between such polymers and the cysteine-rich subdomains of mucus glycoproteins, thus building up the mucus gel layer. Most chemical modifications of chitosan are performed at the free amino groups of the glucosamine units. So far, the alkyl thiomers chitosan–cysteine, chitosan–thiobutylamidine, chitosan–thioglycolic acid, chitosan–N-acetylcysteine, and chitosan–thioethylamidine and the aryl thiomers chitosan–6-mercaptonicotinic acid and chitosan–4-mercaptobenzoic acid have been generated. Due to the immobilization of thiol groups on the chitosan backbone, its mucoadhesive, permeation enhancing, in situ gelling, efflux pump inhibitory, and controlled drug release properties are improved. The great benefits of this new generation of chitosans in comparison to the corresponding unmodified polymers has been verified via numerous in vivo studies on various mucosal membranes. A proof of concept for oral, nasal and buccal drug delivery is provided. This chapter includes an overview of the mechanism of adhesion and the design of thiomers as well as of delivery systems comprising thiolated chitosans and their in vivo performance.