For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single‐strand conformation polymorphism (PCR‐SSCP) analysis. Mutations in exons 5–8 of the p53 gene were investigated by FCR‐SSCF analysis using 103 sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR‐SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR‐SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.