The study performed daily large-scale glycome, transcriptome, and proteome profiling of developing fibers from the two cultivated cotton species, Gossypium barbadense and G. hirsutum, across primary and secondary cell wall stages. It identified delayed cellulose accumulation and distinct compositions of xyloglucans, homogalacturonans, rhamnogalacturonan‑I, and heteroxylans in G. barbadense, along with higher expression of specific glycosyltransferases and expansins, suggesting these molecular differences underlie the superior fiber length and strength of G. barbadense.