Esophageal squamous cell carcinoma (SCC) is one of the most common fatal carcinomas worldwide and has some of the most malignant characteristics among gastrointestinal tumors. Although a high frequency of loss of heterozygosity (LOH) for various genes has been observed in esophageal SCCs, these findings do not provide any information regarding the genetic pathways that may underlie the development and progression of this type of tumor. To clarify the temporal and topographic pathways in the genetic evolution of esophageal SCC, we microdissected multiple foci from superficial mucosal invasive foci of tumors. We then carried out LOH analyses of the microdissected neoplastic foci. Sixteen superficial esophageal SCCs were examined. Three to six carcinoma foci from each superficial esophageal SCC were individually microdissected. We used 12 oligonucleotide primer pairs specific for the microsatellite markers for which frequent LOH in esophageal SCC has been reported. All tumors exhibited LOH of at least three microsatellite loci. A frequent homogeneous LOH pattern was detected for TP53 (60%), D16S518 (43%) and D3S1234 (29%), suggesting that the loss of these alleles is an early event in the development of esophageal SCC. A heterogeneous LOH pattern was detected for D13S325 (87%), D10S559 (73%), D3S1568 (58%), D3S1234 (57%) and D3S1621 (56%), suggesting that the loss of these alleles is a late event in the development of esophageal SCC. All tumors showed the LOH pattern of single clonal neoplasms with genetic progression and divergence. In conclusion, by extensive sampling of SCC lesions with microdissection and LOH analysis of multiple chromosomal loci, we successfully demonstrated dynamic and successive accumulation of genetic alterations in early SCC.