To identify a novel amplified cancer gene a systematic screen of 975 human cancer DNA samples, 750 cell lines and 225 primary tumors, using the Affymetrix 10K SNP microarray was undertaken. The screen identified 193 amplicons. A previously uncharacterized amplicon located on 6p21.2 whose 1 Mb minimal common amplified region contained eight genes (GLO1, DNAH8, GLP1R, C6orf64, KCNK5, KCNK17, KCNK16, and C6orf102) was further investigated to determine which gene(s) are the biological targets of this amplicon. Real time quantitative PCR (qPCR) analysis of all amplicon 6p21.2 genes in 618 human cancer cell lines identified GLO1, encoding glyoxalase 1, to be the most frequently amplified gene [twofold or greater amplification in 8.4% (49/536) of cancers]. Also the association between amplification and overexpression was greatest for GLO1. RNAi knockdown of GLO1 had the greatest and most consistent impact on cell accumulation and apoptosis. Cell lines with GLO1 amplification were more sensitive to inhibition of GLO1 by bromobenzylglutathione cyclopentyl diester (BBGC). Subsequent qPCR of 520 primary tumor samples identified twofold and greater amplification of GLO1 in 8/37 (22%) of breast, 12/71 (17%) of sarcomas, 6/53 (11.3%) of nonsmall cell lung, 2/23 (8.7%) of bladder, 6/93 (6.5%) of renal and 5/83 (6%) of gastric cancers. Amplification of GLO1 was rare in colon cancer (1/35) and glioma (1/94). Collectively the results indicate that GLO1 is at least one of the targets of gene amplification on 6p21.2 and may represent a useful target for therapy in cancers with GLO1 amplification.