Genetic variability in a putative virulence factor, the neutral trehalase (Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., K-m V-max and R-cat). Finally the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower K-cat/K-m. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.