By using the method of PCR-based cDNA library screening, two beta-mannosidase clones, GhManA1 and GhManA2, had been isolated. GhManA1 had a length of 2692 bp coding for a polypeptide of 834 amino acids, and GhManA2 was 3209 bp which encoded a polypeptide of 976 amino acids. GhManA1 and GhManA2 shared an identical sequence of 747 amino acids in their carboxyl-terminals, but were distinctly different in their amino-terminals. Both beta-mannosidases were members of glycosyl hydrolase family 2, which had two conserved glutamine residues in their sequences as the acid-base catalyst and nucleophilic group, respectively. Most surprisingly, the first 93 amino acids in the amino-terminal of GhManA1 was highly homologous to the beta-barrel domain of ATP synthase alpha-/beta-subunit, but an analogous domain has never been found in the sequence of other non-ATP synthase protein. GhManA1 was constitutively expressed in different cotton tissues, and GhManA2 was specifically expressed in fiber cells.