Human papillomavirus type 16 (HPV16) is the most common cause of cervical cancer, but most infections are transient and with lesions not progressing to cancer. There is a lack of specific biomarkers for diagnosis and risk stratification. This study aimed to explore the intra-host HPV16 genomic variation in longitudinal samples from HPV16-infected women with different cervical lesion severity (normal, low-grade, and high-grade). The TaME-seq deep sequencing protocol was used to generate whole genome HPV16 sequences of 102 samples collected over time from 40 individuals. Single nucleotide variants (SNVs) and intra-host single nucleotide variants (iSNVs) were identified in the viral genomes. A majority of individuals had a unique set of SNVs and these SNVs were stable over time. Overall, the number of iSNVs and APOBEC3-induced iSNVs were significantly lower in high-grade relative to normal and low-grade samples, respectively. A significant increase in the number of APOBEC3-induced iSNVs over time was observed for normal samples when compared to high-grade. Our results provide new insight into the dynamics of HPV16 within-hosts evolution. Low number of iSNVs and APOBEC3-induced iSNVs, characteristics of high-grade lesions, could potentially serve as biomarkers to guide triage of HPV-induced cervical precancerous lesions.