Colletotrichum gloeosporionides is the causative agent of a major disease affecting Psidium guajava fruits from fructescence to post-harvest. A polymerase chain reaction (PCR)-based method for the detection of C. gloeosporioides was developed in this study. A set of two primers (CGF and CGR) specific for C. gloeosporioides were designed based on the sequences of the ribosomal internal transcribed spacer (ITS) regions. The specificity of primers was analyzed by the absence of amplified product with DNA of tested isolates. The designed primers amplified a single product of ~390 bp with DNA extracted from isolates of C. gloeosporioides, and did not amplify DNA from several Colletotrichum species and other different fungal genera. The sensitivity of conventional PCR was 10 pg purified genomic DNA in 25 μL reaction. A nested PCR system was established to combine specific and universal ITS primers (ITS1/ITS4), and this enhanced the sensitivity to 1 fg of DNA per 25 μL reaction, representing a 10,000-fold improvement over conventional PCR. The optimised reaction system could detect C. gloeosporionides successfully from diseased plant tissue, infected soil and water. The developed system can detect C. gloeosporioides during the early stages of disease onset and provide evidence to guide management and decision-making.