cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis was used to investigate the differentially expressed genes between sterile and fertile plants of ms5ms6 double-recessive genic male sterility (GMS) two-type line cotton (Gossypium hirsutum L.) at different stages, i.e., sporogenous cell stage, pollen mother cell (PMC) stage, and pollen grain stage. Seventeen differentially expressed fragments were identified. Functional analysis indicated that their corresponding genes may participate in the processes of signal transduction, transcription, energy metabolism, and plant cell wall development. Northern blot demonstrated the credibility of the result of cDNA-AFLP. A sterility restorer factor-like gene, which only expressed in fertile anther and was notably homologous to T cytoplasm male sterility restorer factor 2 of maize (Zea mays L.), was identified in this research.