Abundant research has been conducted on the physiological, biochemical, and anatomical aspects of bamboo culm wall thickening, but its molecular mechanism has not yet been investigated. In this study, we performed whole-genome resequencing of Phyllostachys edulis ‘Pachyloen’, Phyllostachys nidularia f. farcta, Phyllostachys heteroclada f. solida with significantly thicker culm walls, and Schizostachyum dumetorum var. xinwuense with extremely thin culm walls. Moreover, we pioneered the innovative use of gene set subtraction to explore candidate genes that regulate bamboo culm wall thickening. A candidate gene set, containing 633 genes, was obtained by eliminating shared genes that help maintain physiological processes after alignment with the P. edulis reference genome. Starch and sucrose, oxidative phosphorylation, and ribosome were the three most important pathways enriched by differentially expressed genes. Although it cannot be used for hyperfine localization of bamboo wall thickness-regulatory genes, gene set reduction narrows down the range of candidate genes at minimal cost and provides new clues for the application of bioinformatics in plant research.