Abstract Background The development of high-resolution anoscopy (HRA) has advanced our ability to detect anal dysplasia. Historically, HRA is performed in a clinical setting and subsequent ablation is performed in the clinical setting or operating room. The aim of this study was to determine the most effective venue for the performance of HRA. Methods Following institutional review board (IRB) approval, the correlation between anal cytology and HRA performed in the clinic versus in the operating room was evaluated. Data were extracted from our IRB-approved prospective HRA database over the time period of 2013–2017. Results One hundred twenty-eight HRAs were compared (101 in the clinical setting, 27 in the operating room). There was a statistically significant difference in the correlation between anal cytology and HRA pathology for procedures performed in the clinical setting (55% [56/101]) versus those performed in the operating room (82% [22/27]) (p = 0.014). More biopsies were obtained in the operating room than in the clinic setting (3 vs. 1, p < 0.0001). The majority of patients who had HRA in a clinical setting with subsequent HRA in the operating room stated that they preferred to have their HRAs performed in the operating room due to discomfort from the HRA procedure. Conclusions Detection rates for anal dysplasia on HRA, are significantly higher when performed in the operating room. To prevent discomfort in the clinical setting, patients with high-grade dysplasia on anal pap testing may benefit from proceeding directly to the operating room for concurrent HRA and ablation.