Cotton fiber is an ideal model to study cell elongation and cell wall construction in plants. During fiber development, some genes and proteins have been reported to be specifically or preferentially expressed. Mapping of them will reveal the genomic distribution of these genes, and will facilitate selection in cotton breeding. Based on previous reports, we designed 331 gene primers and 164 protein primers, and used single-strand conformation polymorphism (SSCP) to map and integrate them into our interspecific BC1 linkage map. This resulted in the mapping of 57 loci representing 51 genes or proteins on 22 chromosomes. For those three markers which were tightly linked with quantitative trait loci (QTLs), the QTL functions obtained in this study and gene functions reported in previous reports were consistent. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of 52 polymorphic functional primers showed that 21 gene primers and 17 protein primers had differential expression between Emian22 (Gossypium hirsutum) and 3–79 (G. barbadense). Both RT-PCR and quantitative real-time PCR (qRT-PCR) analyses of the three markers tightly linked with QTLs were consistent with QTL analysis and field experiments. Gene Ontology (GO) categorization revealed that almost all 51 mapped genes belonged to multiple categories that contribute to fiber development, indicating that fiber development is a complex process regulated by various genes. These 51 genes were all specifically or preferentially expressed during fiber cell elongation and secondary wall biosynthesis. Therefore, these functional gene-related markers would be beneficial for the genetic improvement of cotton fiber length and strength.