The Entomopoxvirinae or entomopoxviruses (EPVs) infect insects and are used as biological control agents against certain pest species. Although > 40 EPVs across the three major EPV Genera have been described, only the genomes of Amsacta moorei EPV and Melanoplus sanguinepes EPV of the Betaentomopoxvirus Genus have been sequenced. Fewer than 10 genes of the beetle-infecting Alphaentomopoxvirus and NONE of the Diptera (fly)-infecting Gammaentomopoxvirus Genus (mosquitos, chironomids) have been sequenced. Our laboratory has described the Diachasmimorpha longicaudata entomopoxvirus (DlEPV), the first EPV infecting a cyclorraphous dipteran, and have sequenced several genes from its 250-300 kb genome. Unfortunately, there are no sequences from any member of the Gammaentomopoxvirus to facilitate comparison with those from DlEPV and inform its placement within the subfamily. Consequently we have purified a putative EPV (pChEPV) of ~400 nm in diameter, from field-collected infected chironomid larvae and determined the relationship of its proteins to those of DlEPV using anti-DlEPV polyclonal sera to probe western blots. Primers designed from DlEPV-specific genes such as the rifampicin resistance gene (rif), were also used to amplify the pChEPV DNA. The anti-DlEPV sera hybridized with several pChEPV proteins of 29 to ~ 95 kD and a 534 bp fragment was amplified by the DlEPV-specific rif primers. The pChEPV sequence shared >80% amino acid identity with the corresponding rif sequence of DlEPV. Thus, our results provide the first evidence that DlEPV is related to the pChEPV, a likely Gammaentomopoxvirus.