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Chitin synthase (CHS) is a large transmembrane enzyme that polymerizes Uridine diphosphate <i>N</i>-acetylglucosamine into chitin. The genomes of insects often encode two chitin synthases, CHS1 and CHS2. Their functional roles have been investigated in several insects: CHS1 is mainly responsible for synthesizing chitin in the cuticle and CHS2 in the midgut. <i>Lepeophtheirus salmonis</i> is an ectoparasitic copepod on salmonid fish, which causes significant economic losses in aquaculture. In the present study, the tissue-specific localization, expression, and functional role of <i>L. salmonis</i> chitin synthases, <i>LsCHS1</i> and <i>LsCHS2</i>, were investigated. The expressions of <i>LsCHS1</i> and <i>LsCHS2</i> were found in oocytes, ovaries, intestine, and integument. Wheat germ agglutinin (WGA) chitin staining signals were detected in ovaries, oocytes, intestine, cuticle, and intestine in adult female <i>L. salmonis</i>. The functional roles of the <i>Ls</i>CHSs were investigated using RNA interference (RNAi) to silence the expression of <i>LsCHS1</i> and <i>LsCHS2</i>. Knockdown of <i>LsCHS1</i> in pre-adult I lice resulted in lethal phenotypes with cuticle deformation and deformation of ovaries and oocytes in adult lice. RNAi knockdown of <i>LsCHS2</i> in adult female <i>L. salmonis</i> affected digestion, damaged the gut microvilli, reduced muscular tissues around the gut, and affected offspring. The results demonstrate that both <i>Ls</i>CHS1 and <i>Ls</i>CHS2 are important for the survival and reproduction in <i>L. salmonis</i>.