Background: Significant variation is reported in the diagnosis of HPV-associated AIN. We previously observed that bandlike positivity for p16 in >90% of contiguous cells coupled with Ki67 positivity in >50% of lesional cells is strongly associated with high grade AIN. This study was undertaken to determine if addition of p16 and Ki67 immunostaining would reduce inter- and intraobserver variability in diagnosis and grading of AIN.Design: H&E stained slides of 60 anal biopsies were reviewed by three pathologists and consensus diagnoses were achieved: 25 negative, 12 low (condyloma and/or AIN I) and 23 high (9 AIN II and 14 AIN III) grade lesions. The H&E stained slides were diagnosed independently by three additional (“participant”) pathologists. Several weeks later they re-examined these slides in conjunction with corresponding p16 and Ki67 immunostains.Results: Addition of p16 and Ki67 immunostains reduced intra- and interobserver variability, improved concurrence with consensus diagnoses and reduced two-step differences in diagnosis. Negative and high grade AIN diagnoses showed the most improvement in concurrence levels.Conclusion: Addition of p16 and Ki67 immunostains is helpful in the diagnosis and grading of AIN.