To assess the extent of genetic variability of rDNA intergenic spacer (IGS) in Metarhizium sp., 34 strains (27 isolated in Brazil) were sequenced and analyzed together with an additional 20 Metarhizium anisopliae var. anisopliae sequences retrieved from GenBank. Overall, the global nucleotide diversity for the region under study was of 0.090, while for the Brazilian isolates it was only 0.016. Phylogenetic analyses showed four well-supported groups (A, B, C, and D), one of which (D) has not been previously identified. All but one of the Brazilian strains cluster in this novel D phylogroup, suggesting that the genetic variation found in Brazil is a subset of the worldwide M. anisopiliae var. anisopliae variation.Key words: intergenic spacer, Metarhizium, rDNA. The entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina, Hyphomycetes) is an "obligate killer" (Ebert and Weisser, 1997) which has consequently been much studied for potential use as a biological control agent of insect pests in many countries. The utilization and the improvement of mycoinsecticides could be an alternative tool to overcome the considerable environmental impact and high economical costs posed by conventional insecticides (Roberts and St. Leger, 2004). The non-transcribed intergenic region (IGS) and the internal transcribed spacers (ITS) located in the nuclear ribosomal DNA (rDNA) gene cluster evolve rapidly and have been widely used as a tool to evaluate the genetic diversity within populations of fungi (Mavridou and Typas, 1998;Pantou et al., 2003;Hughes et al., 2004;Velásquez et al., 2007) and to resolve problems regarding the identification of species and isolates based on morphological characters (Milner et al., 1994;Driver et al., 2000;Iwen et al., 2002).Even though Metarhizium strains have been used in Brazil against a number of insect pests since the 1980s (Garcia et al., 1984), an evaluation on the intraspecific genetic variability of Metarhizium isolates from Brazil has not so far been performed. Since better characterization of the genetic variation within an entomopathogenic fungus population could be useful for the selection of more efficient isolates for biological control, the aim of this study was to assess the extent of genetic variability in the IGS region of Metarhizium strains with special attention placed on the Brazilian isolates.The 34 Metarhizium sp. isolates (27 isolated in Brazil) that were used in this study (Table 1) were provided by the following organizations: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária -Embrapa) Genetics and Biotechnology Research unit (Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia -CENARGEN), Brasilia-DF, Brazil; School of Biological Sciences, University of Wales, University College Swansea, Swansea, Wales, UK; and Escola Superior de Agricultura Luiz de Queiroz (ESALQ) Brazil. The Isolates were cultivated in liquid complete Cove's medium (CCM) at 28°C for 7 days and DNA was extracted and ...