Betapapillomavirus (betaPV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an association with cutaneous squamous cell carcinoma (SCC). Little is known about the natural history of betaPV infection and the aspects of infection that drive antibody responses. To investigate the relationship between these markers, this study assessed whether the presence or persistence of betaPV DNA in eyebrow hairs and L1 antibodies of the same betaPV type co-occurred more frequently than would be expected by chance in both a cross-sectional assessment and a longitudinal study. betaPV DNA in plucked eyebrow hairs and L1 antibodies in serum were measured in 416 participants of the Australian community-based Nambour Skin Cancer Study in 1996. Similar data were available for a subset of 148 participants in 2003. Observed co-occurrence of betaPV DNA and antibodies was compared with expected values based on prevalence. A case-wise concordance index was used to calculate the overall concordance of betaPV DNA and antibodies of the same type. No significant associations were found between the presence or persistence of betaPV DNA and antibody responses. The age and sex of the host did not influence the association, and nor did SCC status or a history of sunburns. It was concluded that betaPV antibody responses in adults are not primarily driven by betaPV infection as measured in eyebrow hairs. Other factors, such as viral load, may play a more pivotal role in the induction of detectable seroresponses.