Objective: The aim of this study was to evaluate the prevalence of Adenovirus as a etiologic agent of conjunctivitis on a ophthalmic clinic in Viçosa, Minas Gerais, Brazil. Methods: Samples of conjunctival secretion from 91 patients clinically diagnosed with conjunctivitis were subjected to polymerase chain reaction (PCR) using degenerate primers targeted to the gene encoding the structural protein II. Positive samples were subsequently subjected to sequencing and genotyping. Results: PCR results showed 36.3% prevalence of Adenovirus. No differences between the sexes and was found to be higher in the age group 26-65 years with 60.60% of the positive cases. Sequencing of positive cases showed the presence of Adenovirus serotypes 3, 4, 7, 8, and 34 circulating in the region. Conclusion: In Viçosa two in five cases of conjunctivitis has Adenovirus as etiologic agent.