MOTIVATION: Synonymous codon usage bias has been shown to be correlated with many genomic features among different organisms. However, the biological significance of codon bias with respect to gene function and genome organization remains unclear. RESULTS: Guanine and cytosine content at the third codon position (GC3) could be used as a good indicator of codon bias. Here, we used relative GC3 bias values to compare the strength of GC3 bias of genes in human and mouse. We reported, for the first time, that GC3-rich and GC3-poor gene products might have distinct sub-cellular spatial distributions. Moreover, we extended the view of genomic gene domains and identified conserved GC3 biased gene domains along chromosomes. Our results indicated that similar GC3 biased genes might be co-translated in specific spatial regions to share local translational machineries, and that GC3 could be involved in the organization of genome architecture. AVAILABILITY AND IMPLEMENTATION: Source code is available upon request from the authors. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.