Esophageal squamous cell carcinoma (ESCC) is one of the common cancers with a poor prognosis. Incidences of human papillomavirus (HPV) infection range from 0 to 67% in different parts of the world. It has been frequently associated with high-risk HPV genotypes 16 and 18. The present study analyzes the prevalence of HPV infection in ESCC tumor and adjoining mucosa. Fresh tissue samples were obtained from ESCC tumor (group I) and adjoining mucosa (group II). Aliquots of DNA extracts were used. There were 23 patients with paired samples, 19 (83%) were male. HPV was positive in 20/23 (87%). Mean age of HPV positive in group I was 56.63 ± 6.96 and in group II 54.31 ± 7.13 years (P > 0.05). Majority had more than one viral type. HPV52 was the most common observed in 14 (61%) males and two (9%) females. Other common viruses were HPV55, 39, and 59. Smoking had a significant association with viral positivity. p63 and p16 oncoproteins correlated with degree of tumor differentiation but not with viral status. We documented high prevalence of high-risk HPV in ESCC. Our observations support the concept of persistent infection by an oncogenic HPV in cancer development. Our study highlights importance of documenting viral genotype in a defined geographic area.