The objective of this study was to complete the molecular evaluation of five Acacia species including first by determining the genetic diversity of the plants using the polymerase chain reaction (PCR)-based inter-simple sequence repeat (ISSR) method. This investigation was carried out to assess fingerprint and thus genetic variations among the Acacia species. The ISSR method was used to determine DNA fingerprints for Acacia spp. Eight primers were used, with all primers delivering amplification products. Our data show a total of 71 bands of 70 bp to 2,200 bp were amplified, of which 0.77 demonstrated an average polymorphism information content per primer. Among the eight primers tested, the mean annealing temperature was 48°C and average polymorphism information content was between 0.36 and 0.84. The ISSR primers for the five species of Acacia showed four main groups, with a higher level of similarity between these species. These results indicated ISSR markers provide an efficient alternate for identification via DNA fingerprinting of the genetic relationships in Acacia. PCR-based ISSR represents a powerful method that can provide practical information for the development of molecular markers, molecular cytogenetic techniques, and DNA Fingerprinting for application in an Acacia spp breeding program.