Human adenoviruses have been used to develop candidate HIV vaccine vectors, but a major obstacle to the use of vectors derived from Ad5 and other common serotypes is the high prevalence of neutralizing antibodies (NAbs) in humans. Prior studies have reported a prevalence of Ad5 between 60% and 70% in Europe and US, and up to 98% in surveyed African and Asian (Thai) tropical countries. However, few studies have reported on the Ad5 prevalence in Chinese populations. In this study, a total of 1,250 healthy adult serum samples from six administratively separate regions of China were screened by high-throughput luciferase-based virus neutralization assays. Results showed that overall 72% of healthy adults were Ad5 seropositive, and 46.4% with baseline Ad5 NAb titers of >200 in China. Comprehensive analysis by geographical region and age showed that the seroprevalence of Ad5 in southern China such as Guangxi was higher than that in other regions. Geographical differences and climate were considered as the major factors affecting the titer levels of Ad5 in healthy adults. In addition, no apparent gender and ethnic difference was found in any group classified according to region, age, or NAb titer. The present study may provide useful insights for the future development of Ad5-based vaccines and gene therapy. J. Med. Virol. 84:1408–1414, 2012. © 2012 Wiley Periodicals, Inc.