Background: One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits. Methods: In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut (Juglans rega) genome. Results: 5 DCL genes, 13 RDR genes, 15 DRB genes, and 15 AGO genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression. Conclusions: In this study, the genome of the Juglandaceae’s DCL, RDR, DRB, and AGO gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.