Identification of genes specific to fiber development would improve the efforts in developing cotton plants with superior fiber quality. Through genetic introgression, 17 interspecific chromosome substitution lines (CS-B lines) of upland cotton in G. hirsutum (TM-1) have been developed and released recently. These substitution lines have TM1 as background and contain either whole chromosome or chromosome arms of G. barbadense (line 3-79) chromosomes. CS-B25 has chromosome 25 from G. barbadense substituted into TM-1 G. hirsutum was reported to show superior fiber properties. In this study, suppression subtraction hybridization (SSH) combined with Affymetrix cotton genome microarray arrays were used to identify differentially expressed genes in CS-B25. An SSH fiber cDNA library was constructed with differentially expressed genes identified in CS-B25. Microarray analysis showed that 23 genes were up-regulated and 9 down-regulated. Majority of these genes were involved in Ethylene signal pathway, Ubiquitin-proteasome pathway and cell wall synthesis.