The peritrophic matrix (PM) is an acellular structure that lines the gut of most insects. It is an attractive target for pest management strategies because of its close involvement in digestive processes and role as a barrier against pathogens and toxins. The purpose of this study was to identify and characterize the genes that translate for principal components of the Reticulitermes flavipes PM. Genes encoding a gut chitin synthase (CHS), two proteins with peritrophin‐A domains, and a chitin deacetylase were identified from an R. flavipes symbiont‐free gut cDNA library, a pyrosequencing study of termite lignocellulose digestion, and a metatranscriptomic analysis of R. flavipes fed on agricultural biomass. Quantitative expression analysis of the identified genes, in the termite digestive tract, revealed that the transcripts coding for a CHS (RfCHSB) and the proteins with peritrophin‐A domains (RfPPAD1 and RfPPAD2) were predominantly expressed in the midgut, suggesting an association with the PM. The peritrophin identity of the RfPPAD2 gene was confirmed by immunodetection of its translated peptide in the midgut and PM. The discovery and characterization of PM components of R. flavipes provides a basis for further investigation of the viability of this structure as a target for candidate termiticides.