Previous studies showed that chromosomal instability was common in esophageal squamous cell carcinoma (ESCC); however, the mechanisms underlying this instability are unknown. Individuals with deficiencies in telomere maintenance are susceptible to enhanced telomere loss during cell proliferation; such deficiencies could result in telomere dysfunction and genomic instability. We investigated the association between genome-wide chromosomal changes in cancer cells and telomere length/attrition in cancer/stroma cells in 47 ESCC patients. Genome-wide detection of loss of heterozygosity was performed using the Affymetrix GeneChip single nucleotide polymorphism arrays. Telomere length was assessed separately for cancer cells, carcinoma-associated fibroblasts (CAF), infiltrative lymphocytes, and adjacent normal epithelial cells by quantitative fluorescent in situ hybridization using paraffin-embedded sections. Telomere length differed significantly among cell types, such that length in infiltrative lymphocytes > CAFs > cancer cells. Shortened telomeres were observed in cancer cells in 44 of 47 (94%) of the tumors examined. Telomere length in CAFs was significantly associated with chromosomal instability on 4q and 13q and lymphocyte telomere length was significantly associated with instability on chromosomal arms 15q. Although telomere length in cancer cells was not associated with chromosome arm instability, telomere attrition in cancer cells, defined as the telomere length in CAFs minus the telomere length in cancer cells, was significantly associated with chromosomal instability on 13q and 15q. This study provides evidence that telomere shortening is a common genetic alteration in ESCC and that chromosome arm instability is related to both telomere attrition in cancer cells and telomere length in tumor stroma cells.