Carbonyl sulfide (COS) is a new fumigant used in phytosanitary treatments. It was developed as a potential alternative to methyl bromide, which is being phased out because of its ozone-depletion properties. To understand the molecular and cellular mechanisms occurring in fungal pathogens in response to COS fumigation, we cloned 510 cDNA fragments of Alternaria alternata (Fr.) Keissler genes that are differentially expressed; these genes were cloned using suppression subtractive hybridization. Changes in the levels of transcripts of 79 fragments were confirmed by microarray analysis and qRT-PCR. Further homology search revealed that they are highly homologous to 41 genes of other fungi, which were related to general metabolism, growth and division, defense, cellular transport, and signal transduction. These results provide an overview of differential expression profiles of A. alternata genes following COS treatment and some new clues about the mechanism of COS fungitoxicity.