Anal squamous cell carcinoma is relatively rare, but its incidence and mortality have been increasing worldwide. While anal cytology is a sensitive cancer screening modality, its specificity is low, and data for concurrent high‐risk human papilloma virus (HR‐HPV) testing are limited. At our institution, anal cancer screening consists of combined anal cytology and high‐risk human papilloma virus (HR‐HPV) testing on all specimens. The aims of the study were to correlate results of atypical cytological diagnoses atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells cannot exclude high grade squamous intraepithelial lesion (ASC‐H) with HR‐HPV testing and determine if co‐testing may potentially influence management. A retrospective search over 24‐months was performed for anal cytology specimens with diagnoses of ASCUS and ASC‐H. Corresponding HR‐HPV (HPV 16/18 and Other‐31/33/35/39/45/51/52/56/58/59/66/68) results were retrieved, and concordance/discordance was recorded. Cytology results were correlated with anal biopsy diagnoses, when available. A total of 139 patients, including 127 with ASCUS and 12 with ASC‐H, were identified. Of the ASCUS cases, 90/127 (70.9%) had HR‐HPV, and a squamous intraepithelial lesion (SIL) was evident in 20/39 (51.2%) of biopsies. All 12/12 (100%) ASC‐H were associated with HR‐HPV and 3/6 (50%) biopsies had a SIL. Our study supports use of concurrent cytology and HR‐HPV for anal cancer screening cytology. Co‐testing improves specificity of atypical cytology diagnoses and can identify patients requiring further intervention.