Multiple genetic alterations are well recognized as contributing to pancreatic carcinogenesis, although the finding of recurrent copy number changes indicates additional targets remain to be found. The objective of this study was to identify novel targets of genetic alteration that contribute to pancreatic cancer development or progression. We used Representational Oligonucleotide Microarray Analysis (ROMA) to identify copy number changes in pancreatic cancer xenografts, and validated these findings using FISH, quantitative PCR, Western blotting and immunohistochemical labeling. With this approach, we identified a 0.36-Mb amplification at 18q11.2 containing two known genes, GATA-6 and cTAGE1. Using a cutoff value of 3.0 fold compared to haploid controls, copy number gain or amplification was confirmed in 4 of 42 (9.5%) pancreatic carcinomas analyzed. Combined genetic and transcriptional analyses showed consistent overexpression of GATA-6 in all carcinomas with 18q11.2 gain, as well as in the majority of pancreatic cancers examined (17 of 30 cancers, 56.7%) that did not have gain of this region. By contrast, overexpression of cTAGE1 was rare in these same cancers suggesting GATA-6 is the true target of this copy number increase. GATA-6 mRNA overexpression corresponded to robust nuclear protein expression in cancer cell lines and resected tissues consistent with its role as a transcription factor. Intense nuclear labeling was significantly increased in PanIN-3 lesions and infiltrating carcinomas compared to normal duct epithelium (p < 0.000001 and p < 0.003, respectively). Forced overexpression of GATA6 in MiaPaca2 cells resulted in increased proliferation and growth in soft-agar. Gain and overexpression of the development-related transcription factor GATA-6 may play an important and hitherto unrecognized role in pancreatic carcinogenesis.