The majority of low-grade squamous intraepithelial lesions (LSILs) of the cervix regress spontaneously; however, a small proportion of LSILs progress to high-grade squamous intraepithelial lesion (HSIL) if the lesion is not excised. The guidelines for which patients with LSIL should be treated and which may be followed safely are not well established. The goal of this study was to identify a subgroup of patients with LSIL who may require surgical treatment. We hypothesized that patients with LSILs with marked cytological atypia (LSIL-MA) may be at higher risk for subsequent HSIL. In addition, we were interested in whether LSIL-MA was associated with specific human papillomavirus genotypes. Consecutive patients with biopsy diagnosis of LSIL (n = 30) and LSIL-MA (n = 30) were identified. Marked atypia was defined as 5 or more cells with nuclear enlargement of at least 5 times the size of an intermediate cell nucleus or multinucleation with 5 or more nuclei. Patient follow-up was recorded for up to 24 months. Human papillomavirus genotyping was performed using SPF10 PCR and line probe assay. High-grade squamous intraepithelial lesion on follow-up was significantly more common in patients with LSIL-MA (36%) than in patients with LSIL (7%), and negative follow-up was significantly more common in patients with LSIL (50%) than LSIL-MA (23%). Cases of LSIL and LSIL-MA showed similar spectrum of human papillomavirus genotypes. Marked cytological atypia in LSILs identifies a subset of patients with a high rate of HSIL on follow-up. In such patients, an excisional cone biopsy should be strongly considered.