Mass releases of Trichogramma confusum Viggiani and T. maidis Pintureau & Voegele are widely used to control lepidopterous pests. They have long been considered to be the subspecies of T. chilonis Ishii and T. brassicae Bezdenko, respectively. To re-examine the taxonomic status of these closely related Trichogramma species, the internally transcribed spacer 2 (ITS2) of ribosomal DNA was used as a molecular marker to detect between-species differences. The ITS2 regions of 7 different Trichogramma species collected from China, Germany and France were sequenced and the inter-species distances were calculated. To quantify within-species sequence variation, the ITS2 regions of 6 geographical populations of T. dendrolimi Matsumura collected from across China were sequenced and compared. The results show that the ITS2 sequences of T. confusum and T. maidis are sufficiently different from those of T. chilonis and T. brassicae, respectively, that it is difficult to group them as cryptic species, whereas there are only minor differences between the T. dendrolimi populations. The ITS2 sequences identified in this study, coupled with 67 ITS2 sequences from a wide geographical distribution retrieved from GenBank, were then used for phylogenetic analyses. The results support previous records of minor within-species ITS2 sequence divergence and distinct interspecies differences. The cladograms show the T. maidis sequence clustered within T. evanescens Westwood, while the ITS2 sequences of T. confusum and T. chilonis are clustered in different branches. Taken together, these data suggest that T. maidis is not T. brassicae, but a cryptic or sibling species of T. evanescens; T. confusum and T. chilonis are not cryptic species but two closely related sister species.