Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon-carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of beta-hydroxy-alpha-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral beta-hydroxy-alpha-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a L: -threo-phenylserine derivative or L: -threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of L: -threonine or L: -allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of beta-hydroxy-alpha-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.