HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10−7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.Fil: Chouhy, Diego. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas. Área de Virología; ArgentinaFil: Šterbenc, Anja. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Hošnjak, Lea. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Bolatti, Elisa María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas. Área de Virología; ArgentinaFil: Oštrbenk, Anja. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Seme, Katja. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Kocjan, Boštjan J. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Luzar, Boštjan. University of Ljubljana. Faculty of Medicine. Institute of Pathology; SloveniaFil: Giri, Adriana Angélica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas. Área de Virología; ArgentinaFil: Poljak, Mario. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Slovenia