Numerous studies have evaluated human papillomavirus (HPV) DNA load in women, especially HPV-16 viral load, and its role in cervical carcinogenicity. Few studies have examined HPV viral load in men, none among asymptomatic men. The aim of the current study is to quantify HPV-16 viral load in male anogenital specimens and to explore its correlates with anatomic sites. Two-hundred and ninety-four specimens from 42 men who tested positive for HPV-16 at one or more anatomic sites were evaluated. HPV DNA was detected with PGMY 09/11 primer and genotyped with reverse line blot assay followed by HPV-16 viral quantification using type-specific real-time PCR assay (TaqMan). The quantitative PCR assay showed a higher sensitivity in HPV-16 viral DNA detection compared with the reverse line blot assay. Viral load varied significantly by anatomic site (P = 0.019). Penile shaft specimens had significantly higher viral load than any other anatomic site evaluated except for the anal canal. HPV-16 viral load was positively correlated between proximal anatomic sites: perianal and anal canal (P = 0.003), perianal and scrotum (P = 0.011), scrotum and glans/corona (P = 0.045), and scrotum and penile shaft (P = 0.037). In conclusion, the penile shaft seemed to be the preferred site for HPV-16 viral replication. Viral load correlation between proximal sites suggested a possible autoinoculation in male HPV transmission.