The Krüppel-like factor (KLF) family of genes encodes transcriptional regulatory proteins that play roles in differentiation of a diverse set of cells in mammals. For instance, the founding member KLF1 (also known as EKLF) is required for normal globin production in mammals. Five new KLF genes have been isolated from the zebrafish, Danio rerio, and the structure of their products, their genetic map positions, and their expression during development of the zebrafish have been characterized. Three genes closely related to mammalian KLF2 and KLF4 were found, as was an ortholog of mammalian KLF12. A fifth gene, apparently missing from the genome of mammals and closely related to KLF1 and KLF2, was also identified. Analysis demonstrated the existence of novel conserved domains in the N-termini of these proteins. Developmental expression patterns suggest potential roles for these zebrafish genes in diverse processes, including hematopoiesis, blood vessel function, and fin and epidermal development. The studies imply a high degree of functional conservation of the zebrafish genes with their mammalian homologs. These findings further the understanding of the KLF genes in vertebrate development and indicate an ancient role in hematopoiesis for the Krüppel-like factor gene family. © 2001 by The American Society of Hematology.