Inherited dominant polydactylous mice (Mus musculus) was obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis. The mutant mice showed an extra digit on the anterior aspect of one hindlimb that varied in phenotypic severity, and some mice had polydactyly phenotype in both hindlimbs. Alcian blue and alizarin red staining indicated that there was no obvious abnormality of other skeletons in polydactylous heterozygotes except an extra toe in the preaxial of hindlimbs. To determine the position of the mutant gene on the chromosome, microsatellite markers were used to scan the whole genome of the N2 polydactylous mice, which were obtained by back crossbreeding F1 (C57BL/6J ×DBA/2J) with C57BL/6J. Finally, the mutant gene was located between D2mit45 and D2mit184, and Alx4 gene was regarded as the candidate gene of the polydactyly phenotype. Sequence analysis of Alx4 gene for polydactylous heterozygotes revealed an A/T transversion mutation that resulted in an amino acid substitution at position 145 in the protein of a lysine codon (K) by a stop (nonsense) codon.