In the present study, the cDNA sequences of Hsp70 and Hsp90 genes of Isaria farinosa (designated IFHSP70 and IFHSP90) were cloned and characterized using multiple techniques of molecular biology and bioinformatics. The genetic differentiation of the two genes was investigated among 10 geographically separated populations distributed in the Yunnan province. The complete sequence of the IFHSP70 cDNA had a length of 2158 bp, and contained an open reading frame (ORF) of 1962 bp, encoding a 71-kDa polypeptide comprising of 653 amino acids. IFHSP90 cDNA had a length of 2144 bp, and contained an ORF of 2103 bp, encoding a polypeptide of 79.23 kDa, comprising of 700 amino acids. The deduced amino acid sequences of IFHSP70 and IFHSP90 shared high sequence identities with other fungi. Fundamental information pertaining to the protein families, signatures, and conserved motifs of Hsp70 and Hsp90 were also identified. Analysis of molecular variances (AMOVA) from the Hsp70 and Hsp90 genes showed that the genetic variation within-population (83.26%, 83.08%) was greater than among the populations (16.74%, 16.92%). The values of nucleotide diversity (Pi), haplotype diversity (Hd), coefficient of genetic differentiation (Fst), and gene flow (Nm) were calculated. For Hsp70, Pi = 0.0425, Hd = 0.888, Fst = 0.167, Nm = 1.24; For Hsp90, Pi = 0.0420, Hd = 0.894, Fst = 0.169, and Nm = 1.22. These data indicated that the genetic differentiation among 10 different geographical populations of I. farinosa was limited. This study describes, for the first time, cloning, characterization and identification of Isaria farinosa Hsp70 and Hsp90 genes, and provides a preliminary basis for exploring the genetic structure of the genus Isaria using the sequences of Hsp70 and Hsp90 as molecular markers.