<h3>Purpose</h3> To investigate the value of p16 and Ki-67 expression by immunohistochemistry and relation with grade of the anal intraepithelial lesions in HIV-positive patients. <h3>Materials and methods</h3> Twenty HIV-positive patients with high-risk HPV in anal samples underwent to anoscopy and anal biopsy of acetowhite lesions. The p16 and Ki-67 expression by immunohistochemistry using a morphology-independent interpretation p16/Ki-67 dual-stain cytology testing and HPV (human papillomavirus) detection by PapilloCheck testing was analyzed and compared with grade of anal biopsy results. <h3>Results</h3> Anal intraepithelial neoplasia was observed in 7/20 (35%) of the patients (low-grade AIN n=6, and high-grade AIN n=l). Expression of p16/Ki-67 was demonstrated in the sample with high-grade AIN but only 1/6 (16.6%) of the samples diagnosed with low-grade AIN was p16/Ki-67 +. No p16/Ki-67 expression was observed in the negative biopsy sample. In the biopsy sample with high-grade AIN double-stained cells were observed in full thickness of the epithelium and in low-grade AIN the expression were sparse. <h3>Conclusions</h3> By using p16/ Ki-67 staining, despite the presence of multiple high-risk HPV infection and high HPV viral load, the results of this study suggest that the potential for the development of anal high-grade lesion is low.