Abstract Background Amaranthus spp. are problematic weeds and competitors for nutrients in several crops, especially in soybean and corn. Resistance to glyphosate has been detected in several weed species, and a triple mutation in its EPSPS target gene was detected recently in Amaranthus hybridus. Objective The aim of this work was to develop a simple polymerase chain reaction (PCR) method to detect the EPSPS triple mutation in A. hybridus. Methods Two pairs of primers were designed for PCR-based detection of the EPSPS TAP-IVS triple mutation, which confers resistance to glyphosate, in A. hybridus. Results These sets of allele-specific primers were tested on five Amaranthus species and in 65 different field accessions. The PCR reaction using one set of the primers amplifies the wildtype (TAP) allele while the PCR reaction using the other pair of primers amplifies the triple mutation (IVS) allele. The presence of PCR products in both sets of primers identifies the heterozygous resistant individuals, and PCR product amplified only with the triple mutation set of primers identifies the homozygous resistant individuals. A DNA concentration test was performed and the recommend DNA amount to be used is 100 ng. Conclusions We developed and tested two sets of primers to detect the EPSPS TAP-IVS triple mutation and the results showed a 100% genotypic to phenotypic association. The triple mutation detection assay is easy to use and can be applied in a molecular laboratory with basic equipments. Early detection of resistance helps to better manage and control its spreading.