Amino acid sequence analysis of alpha-galactosidases and other proteins of glycoside hydrolase family 27 (GH27) allowed isolation of three major subfamilies, 27a-27c. Unique isomalto-dextranase of Arthrobacter globiformis clustered separately. Eukaryotic proteins formed five clusters on a phylogenetic tree of the family. Bacterial GH27 proteins, which are relatively few, did not form stable clusters. A monophyletic origin of the GH27 family was demonstrated with the use of related proteins of the GH36 family. The structure of the active center and evolution of alpha-galactosidases are discussed.