Background: Few studies have assessed the duration of humoral immunity following yellow fever (YF) vaccination in a non-endemic population. We evaluated seropositivity among U.S. resident travelers based on time post-vaccination. Methods: We identified serum samples from U.S. travelers with YF virus-specific plaque reduction neutralization testing (PRNT) performed at CDC from 1988-2016. Analyses were conducted to assess the effect of time since vaccination on neutralizing antibody titer counts. Results: Among 234 travelers who had neutralizing antibody testing performed on a specimen obtained >/=1 month after vaccination, 13 received multiple YF vaccinations and 221 had one dose of YF vaccine reported. All 13 who received more than one dose of YF vaccine had a positive PRNT regardless of the amount time since most recent vaccination. Among the 221 travelers with one reported dose of YF vaccine, 155 (70%) were vaccinated within 10 years (range 1 month-9 years) and 66 (30%) were vaccinated >/=10 years (range 10-53 years) prior to serum collection. Among the 155 individuals vaccinated <10 years prior to serum collection, 146 (94%) had a positive PRNT compared to 82% (54/66) of individuals vaccinated >/=10 years prior to serum collection (p = 0.01). Post-vaccination PRNT titers showed a time-dependent decrease. Individuals with immunocompromising conditions were less likely to have a positive PRNT (77%) compared to those who were not immunocompromised (92%; p = 0.04). Conclusion: Although the percentage of vaccinees with a positive PRNT and antibody titers decreased over time, a single dose of YF vaccine provided long-lasting protection in the majority of U.S. travelers. A booster dose could be considered for certain travelers who are planning travel to a high risk area based on immune competence and time since vaccination.