Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify β-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as β-amylases. Insights into structural characterization of the oat predicted β-amylases were analyzed using in silico approaches. The identified β-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (β/α)₈-barrel architecture was found in the predicted oat β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)₈-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat β-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.