The non-random amplification of DNA at 9p23 - 24 observed in various types of human cancers, including esophageal squamous cell carcinomas (ESCs), may reflect the locations of important tumor-associated genes. Our previous studies using ESC cell lines defined an amplicon in this region and identified a novel gene, GASC1, as a target of the amplification. Since different regions within the same chromosome arm are often involved in amplification in a syntenic or non-syntenic manner, we characterized the amplicon at 9p23 - 24 in 35 ESC cell lines (29 KYSE series and 6 YES series), and examined possible involvement of non-syntenic amplifications at 9p23 - 24 in 32 primary ESCs. Our results clearly indicated that two target regions for DNA amplification exist at 9p23 - 24; the major amplicon contains GASC1, and the minor one harbors a transcription factor, NFIB, centromeric to the GASC1 locus.