The CCND1-ORAOV1-FGF19-FGF4-FGF3-TMEM16A-FADD-PPFIA1-CTTN (EMS1) locus at human chromosome 11q13.3 is amplified in head and neck tumors, esophageal cancer, Kaposi's sarcoma, bladder tumors, breast cancer, and liver cancer. Fgf4 mRNA is expressed in embryonic stem (ES) cells depending on Sox2 and Pou5f1 (Oct3/Oct4) transcription factors, and in myotomes and limb bud AER depending on MyoD (or Myf5) and GATA transcription factors. Here, rat Fgf3 and Fgf4 complete coding sequences were determined by using bioinformatics. Multiple errors, including one-base insertion and 22-base deletion, were identified within the coding region of rat Fgf4 RefSeq (NM_053809.1 or AB079673.1). Rat Fgf3 and Fgf4 genes, consisting of three exons, were clustered in tail-to-head manner with an interval of about 16 kb. CUTL1 (CCAAT-displacement protein, CDP) and NKX2-5 binding sites and TATA box within 5'-flanking promoter region were conserved among human, rat and mouse Fgf3 orthologs. MYOD and MYOG (Myogenin) binding sites and TATA box within 5'-flanking promoter region as well as GATA, MYOD, SOX2 and POU5F1 binding sites within exon 3 were conserved among mammalian Fgf4 orthologs. Human FGF3 and FGF4 genes were clustered in tail-to-head manner with an interval of about 35 kb. Major repetitive sequence (FGF34Rep1) and minor repetitive sequence (FGF34Rep2) were identified within human FGF3-FGF4 gene cluster. FGF34Rep1 were clustered within the FGF3-FGF4 locus as well as around the IL28RA locus (1p36.11) and the NFAM1 locus (22q13.2). FGF34Rep2 was characterized by the CCA(T/C) repeats. This is the first report on comparative genomics analyses on the Fgf3-Fgf4 locus within human, rat and mouse genomes.