Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a distinct subtype of head and neck cancer. Here, we investigated how frequently brushing remained high-risk (hr)-HPV positive after treatment and whether patients with positive post-treatment brushings have a higher recurrence rate. Following the end of treatment of patients with initially hr-HPV positive OPSCC, surface brushings from the previous tumor site were performed and tested for hr-HPV DNA. Of 62 patients with initially hr-HPV DNA-positive OPSCC, seven patients remained hr-HPV-DNA positive at post-treatment follow-up. Of the seven hr-HPV-positive patients at follow-up, five had a tumor relapse or tumor progression, of whom three died. The majority of patients (55/62) was HPV-negative following treatment. All HPV-negative patients remained free of disease (p = 0.0007). In this study, all patients with recurrence were hr-HPV-positive with the same genotype as that before treatment. In patients who were hr-HPV negative after treatment, no recurrence was observed.