Currently, the pathogenesis of chronic GVHD is unclear. To elucidate the molecular characteristics underlying chronic GVHD, we analyzed the gene expression profiles of 21 mononuclear cell samples from allogeneic hematopoietic stem cell transplantation (HSCT) recipients. Self organizing map (SOM) clustering showed that the entire expression profiles of chronic GVHD samples were clearly different from those of the non-GVHD samples, and significance analysis of microarray (SAM) demonstrated that 120 genes, including PTDSS1, VAV1 and CD3D, were up-regulated, and 5 genes, including calnexin, were down-regulated in GVHD patients. Gene ontology annotation revealed that these genes are related to the phosphorous metabolism and lipid biosynthesis. Quantitative real time polymerase chain reaction (qRT-PCR) experiments validated the up-regulation of PTDSS1, VAV1 and CD3D in separate samples. Pathway-wise global test revealed that differential gene expression in cell cycle and T cell immune-associated pathways were significant between GVHD patients and non-GVHD patients. Seventeen classifier genes selected using a PAM (prediction analysis of microarray) algorithm showed favorable performance (prediction accuracy=0.85) for identifying patients with chronic GVHD. In conclusion, we identified differentially expressed genes and pathways in chronic GVHD patients using microarray analysis, and we also selected diagnostic genes predicting chronic GVHD status.