Codon bias is believed to play an important role in the control of gene expression. In Escherichia coli, some rare codons, which can limit the expression level of exogenous protein, have been defined by gene engineering operations. Previous studies have confirmed the existence of codon pairs preference in many genomes, but the underlying cause of this bias has not been well established. Here we focus on the patterns of rarely-used synonymous codons. A novel method was introduced to identify the rare codons merely by codon pair bias in Escherichia coli.