BackgroundAnal cytology represents a tool for anal cancer screening in high‐risk populations. In addition to accuracy, the reproducibility of the interpretation is of key importance. The authors evaluated the agreement of anal cytologic interpretation between two cytopathologists.MethodsLiquid‐based cytologic slides from human immunodeficiency virus (HIV)‐negative men who have sex with men (MSM) were evaluated by two readers with at least 10 years of expertise in cervical cytology. Cases with a discordant interpretation were reviewed, and a consensus was reached. Human papillomavirus (HPV) genotyping was performed using a proprietary HPV genotyping test. Unweighted and weighted Cohen kappa and 95% confidence interval (CI) values were calculated.ResultsOverall, 713 slides that were adequate for interpretation were evaluated (MSM: median age, 33 years). An HPV test was performed on 620 samples (87.0%). Considering a dichotomous interpretation (negative for intraepithelial lesion or malignancy vs. atypical squamous cells of undetermined significance or worse), the crude agreement between the two readers was 93.3% (kappa = 0.82; 95% CI, 0.77–0.87). Once a consensus for discordant cases was reached, the best agreement was found for the negative for intraepithelial lesion or malignancy category (511 of 528 samples; 96.8%), whereas the atypical squamous cells of undetermined significance category showed the lowest agreement (90 of 117 samples, 76.9%). Considering the individual cytologic categories, overall agreement was 92.1% (kappa = 0.85; 95% CI, 0.81–0.89). The discordant interpretations were not associated with high‐risk HPV infection, HPV16 infection, or MSM age.ConclusionsThe results indicating excellent interobserver agreement in this study substantiate the use of anal cytology in the setting of human immunodeficiency virus‐negative MSM.