To identify the function of β-galactosidase (β-Gal) gene during ripening and softening,we isolated a β-Gal gene,named FaTβgal,from strawberry fruit (Fragaria ananassa Duch.cv Toyonoka) by suppression subtraction hybridization (SSH) and RACE techniques.The full length cDNA sequence of the FaTβgal gene is 2 891 bp and the open reading frame (ORF) is 2 448 bp which encoded a deduced protein including 815 amino acid residues.The FaTβgal protein is a member of the β-Gal gene family of glycoside hydrolase family 35 members,including conservative base sequence “GGPIILSQIENEY” and a galactose binding lectin domain.Blastp analysis revealed 47.1% to 48.1% similarities between FaTβgal and three reported F.ananassa genes of Faβgal1(CAC44500),Faβgal2(CAC44501) and Faβgal3(CAC44502).Phylogenetic analysis showed that FaTβgal was clustered on one independent branch with 24 β-Gal genes of other species.The relative expression of FaTβgal was determined by quantitative real-time PCR (qRT-PCR) during ripening and softening.The results showed that FaTβgal gene was expressed specifically at the mature stage of fruits,and the relative expression levels of FaTβgal genes increased rapidly with fruit ripening and reached a peak at the pink stage,then markedly declined at red fruit stage.The trends of β-Gal expression in two cultivars of Toyonoka and Sweet Charlie were similar during fruit development and ripening.These results indicated that the FaTβgal is a novel gene of the β-Gal gene family,and may play an important role in strawberry fruit ripening and softening.