To identify the molecular alterations found in 30 human papillomavirus (HPV) positive (n = 15) and negative (n = 15) vulvar carcinomas (VC) and their associated preinvasive lesions (VIN [vulvar intraepithelial neoplasia]) and normal epithelium to determine a common molecular pathogenesis of HPV positive and negative VC. Loss of heterozygosity (LOH) at seven 3p chromosomal regions (3p12, 3p14.2, 3p14.3-21.1, 3p21.3, 3p22-24, 3p24.3, 3p25), 13q14 (RB) and 17p13.1 (p53) loci, and TP53 gene mutations in microdissected archival tissues were investigated. Fourteen of fifteen HPV positive VC had HPV 16 DNA sequences. The fractional regional loss index (FRL), an index of total allelic loss at all chromosomal regions analyzed, was greater in the HPV negative VCs than in the HPV positive tumors (FRL = 0.55 versus 0.32; P = .048) and was also greater in the HPV negative high-grade VINs as compared with the HPV positive lesions (0.29 versus 0.02; P = .002). Overall, LOH at any 3p region was frequent (80%) in both groups of cancers and in their associated VIN lesions. Although TP53 gene mutations were present in a minority of VCs (20%), allelic losses at the TP53 locus were frequently present, especially in HPV negative VCs, as compared with the HPV positive tumors (62% versus 15%; P = .02). A greater number of molecular alterations are found in HPV negative VCs compared with HPV positive tumors. Allelic losses at 3p are common early events in vulvar carcinogenesis in HPV negative cancers detected at a high rate in the corresponding high-grade precursor lesions (VIN II/III). TP53 gene mutations with associated 17p13.1 LOH are more common in HPV negative cancers.