The Runx family genes are involved in hematopoiesis, osteogenesis and neuropoiesis, and mutations in these genes have been frequently associated with human hereditary diseases and cancers. Here we report the cDNA cloning of the full Runx gene family of the pufferfish (Fugu rubripes), which comprises frRunx1, frRunx2, frRunx3, frRunt and frCbfb. Fugu is evolutionarily distant from mammals, thus the annotation of the frRunx family genes greatly facilitates comparative genomics approaches. Protein sequence comparison revealed that the fugu genes show high conservation in the Runt domain and PY and VWRPY motifs. frRunx1 had an extra stretch of eight histidine residues, while frRunx2 lacked the poly-glutamine/-alanine stretch that is a hallmark of the mammalian Runx2 genes. Analysis of the promoter regions revealed high conservation of the binding sites for transcription factors, including Runx sites in the P1 promoters. Abundant CpG dinucleotides in the P2 promoter regions were also detected. The expression patterns of the frRunx family genes in various tissues showed high similarity to those of the mammalian Runx genes. The genomic structures of the fugu and mammalian Runx genes are largely conserved except for a split exon 2 in frRunx1 and an extra exon in the C-terminal region of frRunx3 that is missing in mammalian Runx3 genes. The similarities and differences between the Runx family genes of fugu and mammals will improve our understanding of the functions of these proteins.