Epstein-Barr virus (EBV) is known as one of the most widespread oncogenic viruses. Head and neck squamous cell carcinoma (HNSCC) is triggered by various risk factors. The aim of the present study was to determine the EBV infection rate, genotyping and variants frequency in HNSCC patients. In this cross-sectional study, 156 patients with HNSCC were enrolled. Formalin fixed paraffin embedded (FFPE) tissue samples were selected from hospitals affiliated to Iran University of Medical Sciences, Tehran, Iran. The EBV EBNA-3C, EBNA-1 and LMP-1 genes were amplified by PCR and then analyzed and confirmed by nucleotide sequencing. CLC work bench 5, MEGA6 and SPSS v.21 software were used for analysis the raw data. The mean age ± SD (years) of the all patients (n = 156) was 60.5 ± 12.6, in which of 121(77.6%) males it was 60.7 ± 11.9 and of 35 (22.4%) females it was 59.7 ± 14.9. Totally, 20 samples (12.8%) were found to be infected with EBV genome. The EBV genotypes 1 and 2 were calculated 90% (18/20) and 10% (2/20), respectively. vLMP-1 found in 40% (4/10) of all LMP-1 tested samples. Furthermore, the EBNA-1 predominant variants were P-ala followed by P-thr and also there were three P-ala-v2 sub variants. Statistics could not find any significant associations although there were some potentials. By our preliminary study in Iran, it revealed that EBV-1 is the predominant Epstein-Barr virus genotype in head and neck squamous cell carcinoma patients. vLMP-1 isolates showed lower survival rate than others. EBNA-1 variants had no significant association with any specific disease complication.