Recent studies report delayed anti-HIV antibody clearance (seroreversion) among HIV-exposed uninfected infants that may affect diagnostic practices. We evaluated the age-specific seroreversion rates in Thailand. The medical records of HIV-exposed uninfected infants born in January 2000-December 2014 were reviewed. Anti-HIV seroreversion rates at 12, 18 and 24 months were analyzed in 3 periods according to the Thai National Guidelines of prevention of mother-to-child transmission of HIV: zidovudine with or without single dose nevirapine to all women (2000-2006), adding lamivudine plus nevirapine to zidovudine in women with CD4 count <200 cells/mm (2007-2009) and zidovudine plus lamivudine plus boosted lopinavir to all women (2010-2014). In 2013, the serologic test kit was changed from third- to fourth-generation (4G) assay. All the infants were formula fed. Among 736 infants, the overall seroreversion rates at 12, 18 and 24 months of age were 59.38%, 94.57% and 100%, respectively. The seroreversion rates at 12 months of age declined from 68% in 2000-2006 and 65.9% in 2007-2009, to 42.9% in 2010-2014 (P = 0.001). Seroreversion rates at 18 months of age were more than 96.5% before 2013 and decreased to 79.1% in 2013-2014 (P = 0.001) with use of 4G. Multivariate analysis identified antepartum protease inhibitors treatment and the use of 4G testing as independent factors associated with delayed seroreversion. Anti-HIV seroreversion delay in HIV-exposed uninfected infants was associated with use of protease inhibitors and 4G HIV testing, complicating the interpretation to exclude perinatal HIV infection.