The eastern hemlock looper (EHL), Lambdina fiscellaria fiscellaria, the western hemlock looper (WHL), L. fiscellaria lugubrosa, and the western oak looper (WOL), L. fiscellaria somniaria, are closely related insects that are pests in Canadian forests. We have used restriction endonuclease (REN) and Southern blot analyses to characterize DNA of nuclear polyhedrosis viruses (NPVs) isolated from each of the three subspecies of L. fiscellaria. REN analysis of EHL-NPV, WHL-NPV, and WOL-NPV DNAs revealed that they have nonidentical REN-banding patterns. NPV DNA extracted from WOL larvae collected from Douglas fir trees had a different REN pattern than NPV DNA extracted from WOL larvae collected from Garry oak trees. Hybridization of radiolabeled EHL-NPV genomic DNA revealed that the EHL- and WHL-NPVs share considerable DNA sequence similarity, as radiolabeled EHL-NPV DNA hybridized strongly with all WHL-NPV DNA bands. The EHL-NPV polyhedrin gene hybridized strongly with WHL-NPV DNA, but the hybridization patterns of the two NPVs were different. Very weak hybridization was detected between the EHL-NPV polyhedrin gene and DNAs of the two WOL-NPVs. Nucleotide sequence analysis revealed that the polyhedrin genes of EHL-NPV and WHL-NPV differ in sequence by only eight nucleotide substitutions (five third position and three second position changes, resulting in four amino acid substitutions). Phlyogeneitc analysis using the polyhedrin genes of EHL-NPV and WHL-NPV indicate that these two viruses cluster with the Group II NPVs as described by Zanotto, Kessing, and Maruniak (1993, J. Invertebr. Pathol. 62, 147).