The aim of this study was to investigate intratumoral genomic heterogeneity and subclonal structure of esophageal squamous cell carcinoma (ESCC).Multiregion whole-exome sequencing was performed on 24 surgically acquired tumor samples from five untreated ESCC patients collected in 2019 to determine the heterogeneity of mutational landscape within tumors. Phylogenetic analysis and mutation process analysis were used to explore the distribution and dynamic changes of mutation spectrum, and subclone analysis was used to explore the subclonal composition and spatial structure of ESCC.An average of 60.2% of mutations were found heterogenous. TP53 and NOTCH1 mutations were confirmed to be early events, and mutations unique in different tumor regions showed a pattern of branching evolution. A large proportion of mutations were associated with abnormal activity of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family, and significant differences in mutation types between trunk and branch variants were found. Subclonal structure exhibited spatial correspondence and spatial limitations, and different genomic features were characterized between close and distant clones.There is significant intratumoral genomic heterogeneity in the five ESCCs, and their subclonal structure is related to spatial locations.