Bull’s-eye rot caused by Neofabraea vagabunda (= N. alba) is one of the most important postharvest disease of apple, being the only described specie associated with this rot in Chile. The symptoms develop in slow progress, requiring at least three months in cold storage to be visible during postharvest, starting from a preharvest latent infection at the fruit lenticel. Therefore, the aim of this study was to early detect latent infections of N. vagabunda in apple lenticels, using TaqMan probe based qPCR method. The samples analyzed were apple fruits cv. Cripps Pink apparently healthy (n=100); inoculated with N. vagabunda (isolate Nv-UTalca 6.1) (n=50), and with a natural infection developed during cold storage (n=50). The negative controls used were DNA of Diplodia seriata (Ds) and Phacidiopycnis washingtonensis (Pw) isolates obtained from natural infected Chilean apples and DNA of N. kienholzii (Nk) and N. malicorticis (Nm) from pure cultures provided by Wageningen Plant Research. Our results indicated that a qPCR assay was developed and validated to detect DNA sequences specific to N. vagabunda. Consequently, DNA of Ds; Pw; Nk and Nm was not detected using the developed TaqMan probe. Therefore, the early detection of N. vagabunda was positive in fruits apparently healthy (76.2%), natural infected (100%) and N. vagabunda inoculated apples (100%). This method would be also useful for epidemiological studies of Bull’s-eye rot in Chile.