Objective <p>This study examined the association between high risk human papillomavirus viral load and cervical squamous lesions using the Cobas 4800 assay and evaluated its potential as an indicator for identifying CIN2+, and analyzed the impact of multiple infections on viral load and disease risk.</p> Methods <p>A total of 3,838 women who underwent hr-HPV testing were included. The Cobas 4800 assay provided cycle threshold values, which were used as indirect measures of viral load. These values were correlated with histopathological diagnoses obtained within three months of HPV testing.</p> Results <p>Among all participants, 1,660 had normal histology, 900 had CIN1, 1,019 had CIN2/3, and 259 had SCC. HPV16 Ct values decreased with increasing lesion severity in both single and multiple infections, whereas HPV18 and the 12 other hr-HPV types showed no clear correlation with lesion grade. Multiple HPV infections involving HPV16 were not associated with an increased risk of CIN2 + compared with HPV16 single infection. For both HPV16 and HPV18, Ct values did not differ significantly across CIN1, CIN2/3, and SCC between single and multiple infections. ROC curve analysis of HPV16 single infections identified a Ct threshold of 32.25 for identifying CIN2+, with an area under the curve (AUC) of 0.72, a sensitivity of 80.7%, and a specificity of 54.4%.</p> Conclusion <p>HPV16 viral load is correlated with cervical squamous lesions, irrespective of single or multiple infections. HPV16 viral load may serve as a biomarker for indicating cervical squamous lesion occurrence. In this study, no positive association was observed between co-infection and high-grade lesions, and viral loads did not differ significantly between single and multiple infection groups. Integrating viral load assessment into clinical evaluation could thus enable more individualized risk stratification and patient management.</p> Clinical trial number <p>Not applicable.</p>