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Background: Worldwide, transgender women are a high burden population for sexually transmitted diseases. Neovaginal high-risk human papillomavirus (hrHPV) infection has been documented among transgender women, but its prevalence remains unclear. The objective of this study was to determine the prevalence of neovaginal hrHPV in a sample of transgender women in The Netherlands. Methods: Between June 2015 and December 2015, neovaginal samples were obtained from all transgender women who underwent vaginoplasty and attended our outpatient clinic for postoperative follow-up at least 1 year after surgery. High-risk HPV DNA detection and partial genotyping was performed by the HPV-risk assay. Genotyping of non–16/18-hrHPV-positive samples was subsequently performed by GP5+/6+-PCR followed by Luminex suspension array technology. Physical examination and standardized (sexual) history taking was conducted. Results: Valid neovaginal swabs were obtained from 54 transgender women (median age, 40.7 years [range, 19.2–60.3]; median postoperative time, 2.4 years [range, 1.0–34.2]). No transgender women were employed in the commercial sex industry. Of 28 sexually active transgender women, 6 (20%) tested positive for neovaginal hrHPV including types 16, 45, 51, 59, 66, and X. There were no concomitant neovaginal lesions nor neovaginal symptoms. All sexually inactive transgender women tested negative for neovaginal hrHPV. Conclusions: A prevalence of neovaginal hrHPV infection of 20% is observed in Dutch transgender women, who self-reported to be sexually active. The clinical consequences neovaginal hrHPV infection in transgender women require further attention.