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Purpose: The aim of this study was to evaluate the clinical relevance of high-risk human papillomavirus (HR-HPV) negativity at the time of unsatisfactory cervical cytology. Methods: In this retrospective observational study, records were reviewed for patients who had unsatisfactory cervical cytology with HR-HPV testing performed from January 2015 through September 2019 at a large teaching hospital. Pathology results of follow-up cervical cytology and biopsies were assessed. Results: Of 1282 patients with unsatisfactory cervical cytology and negative HR-HPV testing, repeat cytology was negative for intraepithelial lesion (NIL) in 952 (75%) patients, unsatisfactory in 273 (22%) patients, and abnormal in 41 (3%) patients. Median follow-up time was 91 days. The concordance of HR-HPV status between initial unsatisfactory cervical cytology and subsequent satisfactory cervical cytology was 96.3% for HR-HPV negative patients and 68.8% for HR-HPV positive patients. Compared to women who were HR-HPV negative, women who were HR-HPV positive on initial unsatisfactory cytology were at higher risk of subsequent cervical intraepithelial neoplasia (CIN) 2 or greater (odds ratio = 4.91, 95% confidence interval: 1.34–18.03 for E6/E7 mRNA positivity alone; odds ratio = 46.13, 95% confidence interval: 13.45–158.01 for HR-HPV genotype 16 or 18/45 positivity). Conclusion: In the 3 month follow-up of patients with unsatisfactory cervical cytology and negative HR-HPV testing, approximately 3% had abnormal cytology but no cases of HPV related pathology of CIN 2 or greater were found. There was high concordance of negative HR-HPV testing results with those on follow-up satisfactory cervical cytology. [ABSTRACT FROM AUTHOR]