Objective: Anal cytologic testing is being increasingly used as a preventive screening test in high-risk populations. We document anal cytology results, correlating HIV test results, and histopathologic follow-up outcomes from a large integrated health system which recently implemented anal screening. Study Design: Anal Pap tests between May 2007 and August 2009 were studied and correlated with HIV test histories and follow-up histopathologic diagnoses. Results: 688 anal cytologic tests were identified with 7.4% reported as unsatisfactory; 72% of anal cytologic tests were abnormal; 91% of patients were HIV positive. The HIV-positive rate and likelihood of high viral load were both significantly greater among patients with abnormal anal cytology than among patients with negative anal cytology, but did not vary significantly among patients with different categories of abnormal anal cytology. For 459 patients with abnormal anal cytology, 198 had anal biopsies. For patients with abnormal anal cytology findings of ASC-US (atypical squamous cells of undetermined significance), LSIL (low-grade squamous intraepithelial neoplasia), ASC-H (atypical squamous cells, cannot exclude high-grade squamous lesion), and HSIL (high-grade squamous intraepithelial neoplasia), histopathologic intraepithelial neoplasia (AIN)2/3 or 2/3+ diagnoses were established in 46.5, 56.6, 65, and 80.8%, respectively. Conclusions: Patients with any level of abnormal anal cytology result are at significant risk of the presence of histopathologically verifiable high-grade anal intraepithelial lesions. More specific markers for identifying patients at highest risk of progression to invasive anal carcinoma are needed.