Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia.