Background We compared Liquid-Based Cytologic Test (LCT) results diagnosed as atypical squamous cells–cannot exclude high-grade squamous intraepithelial lesion (ASC-H) with high-risk human papillomavirus (hrHPV) infection status and histopathological findings across two cervical screening groups. Additionally, we analyzed the clinical correlation of ASC-H with cervical lesions. Methods This retrospective analysis included 171,688 cases. LCT specimens were divided into two groups: 31,100 from patients who underwent organized cervical screening with artificial intelligence(AI)-assisted interpretation; and 140,588 from patients who underwent opportunistic screening interpreted manually. Of the 663 cases diagnosed with ASC-H, 387 underwent both HPV testing and subsequent histopathological examination. HPV detection methods included 23-type HPV genotyping, and the Cobas 4800 assay which identifies HPV16/18 plus 12 other hrHPV subtypes. Results The overall prevalence of ASC-H was 0.39%. Among 387 paired cases of ASC-H, the total HPV infection rate reached 84.00%, with hrHPV accounting for 97.82% of cases. Women aged <50 years exhibited more single HPV genotype infections and a significantly higher positivity rate for hrHPV than those aged ≥50 years ( p <0.05). In 297 ASC-H samples tested by HPV23 genotyping, the most prevalent hrHPV genotypes were HPV16 (28.62%), HPV52 (21.89%), and HPV58 (17.17%). Among 90 ASC-H samples detected with Cobas 4800 (HPV16/18 plus 12 other hrHPV), the infection rates for HPV16, HPV18, and other 12 hrHPV subtypes were 11.11%, 2.22%, and 82.22%, respectively. Histopathological examination confirmed that 137 of the 387 (35.40%) patients with ASC-H had high-grade squamous intraepithelial lesion or worse (HSIL+), with seven HSIL+ lesions testing negative for HPV. Low-grade squamous intraepithelial lesions (LSIL) were identified in 119 cases (30.75%), including 15 that were negative for HPV. There was no significant difference in HSIL+ positivity observed between the two screening groups, whereas the opportunistic screening group showed a significantly higher positivity rate for LSIL compared with the organized cervical screening group ( p <0.01). Conclusion ASC-H indicates a significant risk of HSIL+, particularly in younger women, regardless of interpretation (AI-assisted or manual). These patients require timely evaluation and suitable treatment, as well as careful follow-up to minimize the risk of missed diagnoses of precancerous lesions and early cervical cancer.