The relationship between human papillomavirus (HPV) and oropharyngeal squamous cell carcinoma has been established. However, data from Ecuador is limited. The objective of this study was to characterize HPV infection in Ecuadorian patients with tongue cancer. Fifty-three patients with tongue cancer treated at the tertiary referral center Sociedad de Lucha Contra el Cancer (SOLCA), Guayaquil, between 2006 and 2011 were identified. Linear Array® HPV genotyping was used to identify the presence and types of HPV on formalin-fixed paraffin-embedded biopsy samples from these patients with tongue cancer. HPV was identified in 42% (n=22) and high-risk (HR) HPV in 17% (n=9), with 18 different HPV types identified. The most common types were the HR HPV 33 (14%) and low-risk HPV 67 (14%), followed by the HR HPV 58. More than one HPV type was identified in 27.3% of cases. HPV 33 was frequently associated with other HPV types. No statistically significant differences in gender (P=0.58) and age (P=0.12) were observed between HPV-positive and HPV-negative cases. HPV was identified in almost half of the tongue cancer samples, with subtypes 33 and 67 being the most common. This suggested that HPV played an important role in this disease in the population studied. Given these results, current HPV vaccines may not be as effective in reducing tongue cancer rates in this population.