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Abstract: Objectives: In a large retrospective study, the association of smoking with human papillomavirus (HPV) genotype and vaginal intraepithelial neoplasia (VAIN) grade was analyzed. Methods: A SNOMED search was performed for vaginal biopsy or resection specimens diagnosed as VAIN over an 11-year period. The diagnosis of VAIN grade was confirmed by histological review. HPV genotype was determined by GP5+/6+ PCR and dot blot hybridization with type-specific oligonucleotide probes. Smoking history was obtained by chart review. Statistical analysis was performed using the chi-square test. Results: We identified specimens from 111 patients (age range 15–84); 64% (n =71) were diagnosed with high-grade VAIN (HGVAIN) and 36% (n =40) with low-grade VAIN (LGVAIN). High-risk (HR) HPV genotypes were identified in 83% of specimens (n =92), other types in 17% (n =19). Twenty-one different HPV genotypes were detected in total. Smoking history was available for 81% (n =90). Forty-one percent (n =37) had a positive smoking history. There was no significant difference in infection with HR vs. other types (p =0.92) among smokers when compared to non-smokers. In patients with HR HPV genotypes, smokers were at an increased risk for HGVAIN lesions when compared to patients who had never smoked (83% vs. 59%, p =0.02). Conclusions: These data indicate an increased risk for HGVAIN in HR HPV positive women who smoke compared to HR HPV positive non-smokers. [Copyright &y& Elsevier]