The complete coding sequence of one pig gene, CDK20, was amplified by RT-PCR. Sequence analysis revealed that the porcine CDK20 gene encodes a protein of 343 amino acids which has high homology with the cyclin-dependent kinase 20 (CDK20) of eight species: cattle (96%), giant panda (96%), mouse (96%), rabbit (96%), rat (96%), dog (95%), human (93%) and Sumatran orangutan (93%). This novel porcine gene was then deposited into the GenBank database and finally assigned to GeneID: 100157041. This gene is structured in eight exons and seven introns as revealed by computer-assisted analysis. Polymorphism analysis revealed that there was a T/C substitution at the position of 96-bp of exon 4 and this led to the amino acid alteration from the Arg residue to the Cys residue. PCR-Alu I-RFLP analysis indicated that eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association analysis showed that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows. Therefore, CDK20 gene could be a useful candidate gene for increasing the litter size of pigs. These data serve as a foundation for further insight into the porcine CDK20 gene.