The mitochondrial cytochrome oxidase I (COI) gene region of Cephus pygmeus (L.) (Hymenoptera: Cephidae) was characterized at the nucleotide and amino acid levels. The partial sequence (687 bp) of the gene was determined and compared with other cephids and insect species at both the nucleic acid and amino acid sequence levels. The examined fragment was placed at an equal distance to both ends of the gene and consisted of 13 structural regions. The 5' end of the partial COI gene was more informative than the 3' end at the intraspecific level. Pairwise sequence divergences ranged from 0.15% to 1.17% at the intraspecific level, while the rate varied from 8.14% to 15.32% in comparison with other Cephus species. The percentage of nucleotide composition at each codon position and the rate of nucleotide substitutions were variable and displayed a bias toward A + T. A strong bias of transitional substitutions was observed at the intraspecific level, but there was a lower rate at the interspecific level. The codon usage patterns and the extent of the substitutions at synonymous codons were also investigated at the amino acid level. These results suggest that the characterized region is informative for estimating both intra- and interspecific relationships due to its possession of both completely conserved and variable regions.