Background: Intratumoral heterogeneity (ITH) is associated with the poor prognosis in a variety of solid tumors, because of the therapeutic failure and drug resistance. However, we still lack understanding of the relationship between ITH and immunotherapy for advanced head and neck squamous cell carcinoma (HNSCC). Methods: We used the Bioconductor R package Maftools to analyze somatic variants of HNSCC from MSK-IMPACT-1611 study cohort (PMID: 30643254) with data from 68 patients. Then, a mutant-allele tumor heterogeneity (MATH) algorithm was used to measure ITH score. Finally, we analyzed the difference in the genomes, and the association with tumor mutational burden (TMB) between the ITH score high (ITH-H) and ITH score low (ITH-L) groups, and the effect of ITH on the prognosis of immunotherapy. Results: There was no significant difference in the high frequency mutated genes between the high ITH and low ITH groups. The genes with the highest mutation frequency in ITH-H group were TP53 (90.48%), TERT (47.62%), CDKN2A (38.1%), FAT1 (33.33%), NOTCH1 (28.57%), KMT2D (19.05%), APC (14.29%), and ASXL1 (14.29%), while the genes with the highest mutation frequency in ITH-L group were TP53 (44.68%), PIK3CA (38.3%), TRET (36.17%), FAT1 (21.28%), NOTCH1 (19.15%), KMT2C (17.02%), KMT2D (17.02%), and CDKN2A (14.89%). The median overall survival (mOS) of ITH-L group was significantly higher than that of ITH-H group (not reach versus 10 months, hazard ratio (HR) = 2.15, 95%CI 1.05−4.4, P = 0.032). There was no significant difference in median TMB levels between ITH-L and ITH-H groups (P = 0.55). The TMB high (TMB-H) patients had no significantly better mOS than the TMB low (TMB-L) group (HR = 0.54, 95%CI 0.25−1.16, P = 0.11). A pooled analysis of ITH and TMB show that the ITH-L+TMB-H group had significantly better improvement mOS than that in the ITH-L+TMB-L, ITH-H+TMB-H, and ITH-H+TMB-L group (HR = 0.71, 95%CI 0.13−1.35, P = 0.048). Conclusions: ITH may serve as a positive predictor of immunotherapy in patients with advanced HNSCC and is independent of TMB.