Fushi tarazu factor 1 (Ftz-F1, NR5A) is a zinc finger transcription factor that belongs to the nuclear receptor superfamily and regulates genes involved in sterol and steroid metabolism in gonads, adrenals, liver, and other tissues. To understand the evolutionary origins and developmental genetic relationships of the Ftz-F1 genes, we have cloned four homologous Ftz-f1 genes in zebrafish, called ff1a, ff1b, ff1c and ff1d. These four genes have different temporal and spatial expression patterns during development, indicating that they have distinct mechanisms of genetic regulation. Among them, the ff1a expression pattern is similar to mammalian Nr5a2, while the ff1b pattern is similar to that of mammalian Nr5a1. Genetic mapping experiments show that these four ff1 genes are located on chromosome segments conserved between the zebrafish and human genomes, indicating common ancestral origin. Phylogenetic and conserved synteny analysis show that ff1a is the ortholog of NR5A2, and that ff1b and ff1d genes are co-orthologs of NR5A1 that arose by a gene duplication event, probably a whole genome duplication, in the ray-fin lineage, and each gene is located next to an NR6A1 co-ortholog as in human, showing that the tandem duplication occurred before the divergence of human and zebrafish lineages. ff1c does not have a mammalian counterpart. Thus we have characterized the phylogenetic relationships, expression patterns, and chromosomal locations of these Ftz-F1 genes, and demonstrated their identities as NR5A genes in relation to the orthologous genes in other species.