The parasitic wasp, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), introduces an entomopoxvirus (DlEPV) into its Caribbean fruit fly host, Anastrepha suspensa. (Loew) (Diptera: Tephritidae), during oviposition. DlEPV has a 250–300 kb unipartite dsDNA genome, that replicates in the cytoplasm of the host's hemocytes, and inhibits the host's encapsulation response. The putative proteins encoded by several DlEPV genes are highly homologous with those of poxviruses, while others appear to be DlEPV specific. Here, a 2.34 kb sequence containing a 1.64 kb DlEPV open reading frame within a cloned 4.5 kb EcoR1 fragment (designated R1–1) is described from a DlEPV EcoRI genomic library. This open reading frame is a homolog of the vaccinia virus rifampicin resistance (rif) gene, D13L, and encodes a putative 546 amino acid protein. The DlEPV rif contains two EcoRV, two HindIII, one XbaI, and one DraII restriction sites, and upstream of the open reading frame the fragment also contains EcoRV, HindII, SpEI, and BsP106 sites. Early poxvirus transcription termination signals (TTTTTnT) occur 236 and 315 nucleotides upstream of the consensus poxvirus late translational start codon (TAAATG) and at 169 nucleotides downstream of the translational stop codon of the rif open reading frame. Southern blot hybridization of HindIII-, EcoRI-, and BamH1-restricted DlEPV genomic DNA probed with the labeled 4.5 kb insert confirmed the fidelity of the DNA and the expected number of fragments appropriate to the restriction endonucleases used. Pairwise comparisons between DlEPV amino acids and those of the Amsacta moorei, Heliothis armigera, and Melanoplus sanguinipes entomopoxviruses, revealed 46, 46, and 45 % similarity (identity substitutions), respectively. Similar values (41–45%) were observed in comparisons with the chordopoxviruses. The mid portion of the DlEPV sequence contained two regions of highest conserved residues similar to those reported for H. armigera entomopoxvirus rifampicin resistance protein. Phylogenetic analysis of the amino acid sequences suggested that DlEPV arose from the same ancestral node as other entomopoxviruses but belongs to a separate clade from those of the grasshopper- infecting M. sanguinipes entomopoxvirus and from the Lepidoptera-infecting (Genus B or Betaentomopoxvirus) A. moorei entomopoxvirus and H. armigera entomopoxvirus. Interestingly, the DlEPV putative protein had only 3–26.4 % similarity with RIF-like homologs/orthologs found in other large DNA non-poxviruses, demonstrating its closer relationship to the Poxviridae. DlEPV remains an unassigned member of the Entomopoxvirinae ( http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm) until its relationship to other diptera-infecting (Gammaentomopoxvirus or Genus C) entomopoxviruses can be verified. The GenBank accession number for the nucleotide sequence data reported in this paper is EF541029.