Cryptococcus neoformans mutants lacking each of the eight putative chitin synthase genes (CHS) have been previously generated. However, it is still unclear how deletion of chitin synthase genes affects the cryptococcal capsule. Since the connections between chitin metabolism and capsular polysaccharides in C. neoformans are numerous, we analyzed the effects of deletion of CHS genes on capsular and capsule-related structures of C. neoformans. CHS deletion affected capsular morphology in multiple ways, as determined by scanning electron microscopy and immunofluorescence analysis. Molecular diameter, serological reactivity and export of capsular polysaccharide were also affected in most of the chsΔ mutants, but the most prominent alterations were observed in the chs3Δ strain. C. neoformans cells lacking CHS genes also had altered formation of extracellular vesicles and variable chitinase activity under stress conditions. These results reveal previously unknown functions of CHS genes that greatly impact the physiology of C. neoformans.2022-01-01