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Objective: To evaluate the rate and types of human papillomavirus infection in vulvar intraepithelial neoplasia. Methods: We detected and identified HPV in 40 VIN cases with 67 lesions using PCR based reverse line blot hybridization and DNA sequencing. Among the 40 patients, 13 were diagnosed as VIN Ⅲ, 11 as VIN Ⅱ, and 16 as VIN Ⅰ. 31 patients had multifocal disease. First a fragment of 150 bp was amplified from the L1 region of HPV with GP5/GP6 primers. If the result was negative, a short fragment of 65 bp was amplified also from the L1 region with SPF1/SPF2 primers. Results: Using general primer GP5/GP6, the positive rate was 52.2% (35/67). Using a short PCR fragment (SPF PCR), the positive rate of the rest 32 lesions was 81.2% (26/32). The total positive rate was 91.0% (61/67). 90% of the HPV types found in VIN were high risk types. All 35 GP PCR products were analyzed by sequencing. The gene types of 31 mono-infection lesions were in accordance with the reverse line blot results, while sequence results of the 4 multi-infection samples could not be analyzed. The SPF PCR products were also sequenced, 24 of the 26 SPF PCR products could be analyzed and 2 samples failed. 80.6% (25/31) cases with multifocal VIN displayed the identical type of HPV, suggesting monoclonality in different lesions from the same patient. Conclusion: The high risk type of HPV is associated with vulvar intraepithelial neoplasia and may be necessary for development of HPV-associated invasive vulvar carcinoma.