AbstractTo inform optimal approaches for detecting anal precancers, we performed a systematic review and meta‐analysis of the diagnostic accuracy of anal cancer screening tests in different populations with elevated risk for anal cancer. We conducted a literature search of studies evaluating tests for anal precancer and cancer (anal intraepithelial neoplasia grade 2 or worse, AIN2+) published between January 1, 1997 to September 30, 2021 in PubMed and Embase. Titles and abstracts were screened for inclusion and included articles underwent full‐text review, data abstraction and quality assessment. We estimated the prevalence of AIN2+ and calculated summary estimates and 95% confidence intervals (CI) of test positivity, sensitivity and specificity and predictive values of various testing strategies, overall and among population subgroups. A total of 39 articles were included. The prevalence of AIN2+ was 20% (95% CI, 17‐29%), and ranged from 22% in men who have sex with men (MSM) living with HIV to 13% in women and 12% in MSM without HIV. The sensitivity and specificity of cytology and HPV testing were 81% and 62% and 92% and 42%, respectively, and 93% and 33%, respectively for cytology and HPV co‐testing. AIN2+ risks were similar among those testing positive for cytology, HPV, or co‐testing. Limited data on other biomarkers (HPV E6/E7 mRNA and p16/Ki‐67 dual stain), suggested higher specificity, but lower sensitivity compared with anal cytology and HPV. Our findings provide important evidence for the development of clinical guidelines using anal cytology and HPV testing for anal cancer screening.