In previous studies, we had demonstrated that allelic losses in esophageal cancer (EC) tissues are frequently involved in chromosomes 3 and 9 and that EC patients and their blood relatives have low capacity to repair damaged DNA and showed genetic instability. To better define the deleted chromosomal loci and understand the genetic instability in EC tissues, we selected 12 microsatellite markers (D3S1232, D3S1238, D3S1289, D3S1480, D3S647, D3S966, D3S1317, D3S659, D9S156, D9S171, D9S176 and GSN) to examine 36 paired EC tissues for loss of heterozygosity (LOH) and microsatellite instability (MIN) on chromosomes 3 and 9. The frequent LOH was found at D9S156(9p21), D3S647(3p23) and D3S1480(3p14.2), implying the possible existence of tumor suppressor genes near the deleted loci. Higher LOH incidence at D9S156 (9/18) and D3S1480 (8/19) was observed in EC tissues from Beijing, a low EC area. More frequent LOH at D3S647 (6/14) was found in EC tissues from Yangquan, a high EC area. This geographic difference of LOH occurrence was indicative of genetic heterogeneity in the etiology of EC. 24 of 36 (66.7%) EC tissues showed MIN at one or more chromosomal loci. The putative EC suppressor genes on chromosomes 3 and 9 and the molecular basis of the genetic instability associated with EC remain to be elucidated.