In this study, two enolase genes were isolated and characterized from the Chinese oak silkworm, Antheraea perny, which were designated as enolase I and II, respectively. The enolase I cDNA sequence was 1712 bp with an open reading frame (ORF) of 1302 bp encoding 433 amino acids. The enolase II cDNA sequence was 1549 bp with an ORF of 1296 bp encoding 431 amino acids. The amino acid sequences of the two genes share several conserved features/sites of enolase. Antheraea pernyi enolase I shows 93%-97% sequence identity to enolases of lepidopterans available to date, 75%-82% identity to enolases of other invertebrates, 60%-72% identity to enolases of other organisms including vertebrates, plants, and fungi. Antheraea pernyi enolase II shows 84% identity to Bombyx mori enolase II, but 60% identity to A. pernyi enolase I. In the phylogenetic tree, enolase II sequences from A. pernyi and B. mori were clearly separated from the majority of enolase sequences of higher organisms including A. pernyi and B. mori enolase I sequences. By sequence comparisons and phylogenetic analysis, we suggest that enolase II from A. pernyi and B. mori may be a new member of the enolase superfamily. Antheraea pernyi enolase I mRNA was found in all tested tissues whereas enolase II mRNA was expressed specifically in the spermaries and ovaries, suggesting that the product of enolase II gene may be related to reproduction. The transcript abundance of A. pernyi enolase I gene was significantly down-regulated after cold shock and significantly up-regulated after heat shock, suggesting that A. pernyi enolase I gene may be inducible by temperature stress.