A cDNA library construction method was established that allows gene expression of the entomopathogenic fungus Metarhizium anisopliae var. acridum to be studied during its growth on Locusta migratoria wings. Before inoculation, locust wings were treated with an RNA fragmentation buffer to degrade host nucleic acid. Reverse transcription-PCR analysis of several constitutively expressed locust genes was used to confirm the degradation of locust nucleic acid. A normalized cDNA library of M. anisopliae var. acridum germinating and differentiating on locust wings was constructed and analyzed. Analysis of 162 unigenes out of 221 sequenced inserts from the cDNA library revealed that the cDNA library was not contaminated with locust transcripts, and contained previously characterized genes known to be expressed during infection. Further studies of the expressed sequence tags from this library will provide insights into the molecular basis of infection of this fungus.