Diglyphus isaea is one of the primary parasitoids of agromyzid leafminers. Mitochondrial cytochrome oxidase subunit I sequences from 10 Chinese localities were used to investigate geographic variation within this widespread and morphologically uniform parasitoid. Phylogeographic analyses and sequence variation indicated that this species was actually a species-complex. We used an amber fossil record of the genus Entedon (Eulophidae: Entedoninae) as a reference to estimate the divergence time of major clades within D. isaea. With reference to the fossil record of its hosts, our results indicate that associations between parasitoids and leaf-miners could have been possibly established 38.2 million years ago.