Objective(s): Variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer. Materials and Methods: DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of HETEROZYGOSITY (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12. 3), D13S267 (13q12. 3), D9S171 (9p21), D2S123 (2p), D5S2501 (5q21) and TP53 (17p13. 1) analyzed on 6% denaturing polyacrylamide gel electrophoresis. Results: Statistical analysis indicated a near significant reverse correlation between grade and LOH (P= 0. 068, correlation coefficient=-0. 272). Specifically, increased LOH in tumor DNA has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors (P= 0. 002 and P= 0. 016 respectively). In addition, higher number of chromosomal loci with LOH showed a reverse correlation with lymph node metastasis (P= 0. 026, correlation coefficient=-0. 485). Furthermore, there was a positive correlation between addiction and MSI (P= 0. 026, correlation coefficient= 0. 465). Conclusion: Microsatellite DNA alterations may be a prognostic tool for detection and the evolution of prognosis in patients with SCC of esophagus. It can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium, cigarette, water pipe or alcohol can be a susceptibility factor(s) for MSI.