BackgroundThe causal role of high-risk Human papillomavirus (HR-HPV) in the pathogenesis of anogenital cancers is well established. In contrast, information on HR-HPV distribution of continuous anatomic sites within the female genital tract is limited, and the impact of sample type on the clinical performance in HPV-based cervical cancer screening warrants investigation.MethodsA total of 2,646 Chinese women were enrolled in the study from May 2006 to April 2007. We analyzed the infection features by infection status and pathological diagnoses of 489 women with complete HR-HPV type and viral load data on the cervix, upper vagina, lower vagina, and perineum samples. Additionally, we assessed the clinical performance for detecting high-grade cervical intraepithelial neoplasia of grade two or worse (≥ CIN2) among these four types of samples.ResultsHR-HPV positivity rate was lower in the cervix (51.53%) and perineum (55.83%), higher in the upper (65.64%) and lower vagina (64.42%), and increased with the severity of cervical histological lesions (all P<0.001). Single infection was more dominant than multiple infections at each anatomic site of the female genital tract. The proportion of single HR-HPV infection decreased successively from the cervix (67.05%) to the perineum (50.00%) (Ptrend=0.019) in cervical intraepithelial neoplasia grade 1 (CIN1) and was higher in samples of the cervix (85.11%) and perineum (72.34%) in ≥ CIN2. In addition, the highest viral load was observed in the cervix compared to the other three sites. The overall agreement of the cervical and perineum samples was 79.35% and increased continuously from normal (76.55%) to ≥ CIN2 (91.49%). As for the detection of ≥ CIN2, the sensitivity was 100.00%, 97.87%, 95.74%, and 91.49% for the cervix, upper vagina, lower vagina, and perineum samples, respectively.ConclusionsSingle HR-HPV infection predominated throughout the female genital tract, but the viral load was lower compared to multiple HR-HPV infections. Despite the decreasing viral load from cervix to perineum, the clinical performance for detecting ≥ CIN2 of the perineum sample was comparable to that of the cervix.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12985-023-02073-4.