In Nigeria, deforestation has led to an unimaginable loss of genetic variation within tree populations. Regrettably, little is known about the genetic variation of many important indigenous timber species in Nigeria. More so, the specific tools to evaluate the genetic diversity of these timber species are scarce. Therefore, this study developed species-specific markers for Pterygota macrocarpa using state-of-the-art equipment. Leaf samples were collected from Akure Forest Reserve, Ondo State, Nigeria. DNA isolation, quantification, PCR amplification, gel electrophoresis, post-PCR purification, and sequencing were done following a standardized protocol. The melting temperatures (TM) of the DNA fragments range from 57.5 ℃to 60.1 ℃ for primers developed from the MatK gene and 58.7 ℃ to 60.5 ℃ for primers developed from the RuBisCo gene. The characteristics of the ten primers developed are within the range appropriate for genetic diversity assessment. These species-specific primers are therefore recommended for population evaluation of Pterygota macrocarpa in Nigeria.