The insect pathogenic fungus Metarhizium anisopliae is widely used as an insect biocontrol agent. The M. anisopliae conidium plays an important role in pathogenesis and disease transmission. The aim of this study was to identify genes whose expression is up-regulated during conidiogenous cell development. This is a powerful strategy for obtaining insight into the molecular events that regulate conidiation. We isolated genes that are preferentially expressed in the developing conidiophores of the common fungal locust pathogen M. anisopliae CQMa102 using suppression subtractive hybridization. Based on the results of cDNA array dot blotting, we identified 109 unique expressed sequence tags (ESTs) that were up-regulated more than fivefold during conidiophore formation. Among these 109 ESTs were 45 (41.3%) with significant similarity to NCBI annotated hypothetical proteins, 35 (32.1%) with low similarity to known or predicted genes that might represent novel genes, and 29 (26.6%) with significant similarity to known proteins involved in various cell and molecular processes, such as ell structure and function, cell metabolism, protein metabolism, stress response, nucleic acid metabolism, and cell cycle and growth. We confirmed the up-regulation of 11 randomly selected genes with real-time reverse transcriptase-PCR analysis. The results of this study provide a preliminary description of genes that may be involved in the molecular regulation of fungal conidiogenesis.