Fructose 1,6-biphosphate aldolase (FBA) is a key enzyme in plants, which is involved not only in glycolysis and gluconeogenesis in the cytoplasm, but also in the Calvin cycle in plastids. Research on FBAs in various organisms has been reported, but there is none on FBAs in Arabidopsis at the molecular level. In the current study, eight FBA family genes (AtFBA1-8) were identified and analyzed in Arabidopsis thaliana. These genes have a highly conserved aldolase-type TIM barrel domain and a C-terminal peptide, but variable N-terminal peptides. Based on the phylogenetic analysis of FBA protein sequences from Arabidopsis and other plant species, AtFBA family was classified into two subfamilies, including three members (AtFBA1-3) with high similarities to FBAs occurring at plastid, and five (AtFBA4-8) with high similarities to FBAs localized in the cytoplasm. By confocal microscopy analysis with GFP fusion protein, AtFBA3 and AtFBA4 as well as AtFBA6 were observed to be localized in the plastid and cytoplasm, respectively. At least two duplicated gene pairs of AtFBA1 and AtFBA2, as well as AtFBA4 and AtFBA8 were found. Transcript level analysis of AtFBA genes in various tissues revealed the unique and overlapping expression patterns of plastid and cytosol AtFBA genes, suggesting that these genes may function at different stages of plant growth and development. Interestingly, AtFBA1, AtFBA2, AtFBA5 and AtFBA7 showed undetectable expression in roots. The expression patterns of AtFBA genes under different stress conditions suggested that all the members showed different expression patterns in response to stresses, including ABA, NaCl, Cd, abnormal temperature and drought, and, except for AtFBA3, most of the AtFBA genes were significantly responsive to drought stress in roots. Moreover, AtFBA1, AtFBA2, AtFBA5, AtFBA7 and AtFBA8 were induced by at least one of three sugars (sucrose, glucose and fructose) after 24h of treatment. Further functional analyses indicated important clues of AtFBA2, AtFBA6 and AtFBA8 in plant growth, stress responses and development, respectively. Thus these results provide additional knowledge on AtFBA families and their roles.