To identify the chromosomal aberrations associated with the progression of liver cancer, we applied expression imbalance map analysis to gene expression data from 31 hepatocellular carcinomas and 19 noncancerous tissues. Expression imbalance map analysis, which detects mRNA expression imbalance correlated with chromosomal regions, showed that expression gains of 1q21-23 (74%), 8q13-21 (48%), 12q23-24 (41%), 17q12-21(48%), 17q25 (25%), and 20q11 (22%) and losses of 4q13 (48%), 8p12-21 (32%), 13q14 (32%), and 17p13 (29%) were significantly associated with hepatocellular carcinoma. Most regions with altered expression identified by expression imbalance map were also identified in previous reports using comparative genomic hybridization. We demonstrated chromosomal copy number gain in 1q21-23 and loss in 17p13 by genomic quantitative PCR, suggesting that gene expression profiles reflect chromosomal alterations. Furthermore, expression imbalance map analysis revealed that more poorly differentiated hepatocellular carcinoma contain more chromosomal alterations, which are accumulated in a stepwise manner in the course of hepatocellular carcinoma progression: expression imbalance of 1q, 8p, 8q, and 17p occur as early events in hepatocarcinogenesis, and 12q, 17q25 and 20q occur as later events. In particular, expression gain of 17q12-21 and loss of 4q were seen to accumulate constantly through the dedifferentiation process. Our data suggest that gene expression profiles are subject to chromosomal bias and that expression imbalance map can correlate gene expression to gene loci with high resolution and sensitivity.