<b><i>Objective:</i></b> Our main objective was to assess the association between the markers p16 and Ki-67 and recurrence of disease in patients previously treated for cervical high-grade squamous intraepithelial lesion (HSIL). <b><i>Design:</i></b> This is a case-control study at the National Cancer Institute conducted between 2005 and 2015. Of the patients with a pathologically confirmed diagnosis of HSIL, 107 cases were selected. They were divided into 2 groups: 28 cases with recurrence after treatment and a control group of 79 patients without recurrence. We identified clinical, pathological, and treatment variables. <b><i>Methods:</i></b> Two experienced pathologists performed immunohistochemical analysis of biomarkers; they agreed on their interpretation, and we calculated the odds ratios (ORs) associated with recurrence. For group comparisons, we used the Wilcoxon signed-rank, χ², or Fisher’s exact test, depending on the type of variable. We conducted logistic regression models to estimate ORs and determine the factors associated with recurrence. The recurrence-free period was defined as the time frame between conization and either recurrence of disease or the last date the patient was seen. We used Kaplan-Meier plots to visualize survival curves and log-rank tests to compare the curves. We established a <i>p</i> value &#x3c;0.05 as statistically significant. <b><i>Results:</i></b> After pathologists performed immunohistochemical analysis, they achieved an agreement level of 83.7% for p16 and 60% for Ki-67. We did not find an association between recurrence and either p16 expression (<i>p</i> = 0.69) or the percentage of Ki-67 expression (<i>p</i> = 0.71). The recurrence-free period analysis did not reveal a difference in p16 expression (<i>p</i> = 0.57) nor in the percentage of Ki-67 expression in the 3-tiered scale (<i>p</i> = 0.56). <b><i>Limitations:</i></b> Our main limitation was a reduced sample size. <b><i>Conclusion:</i></b> We found no association between p16 and Ki-67 positivity and the risk of recurrence in previously treated HSIL.