This paper describes a diagnostic system to verify interspecific hybrids between Acacia mangium and A. auriculiformis using single nucleotide polymorphism (SNP) markers. Forty-eight DNA fragments were selected based on random amplified polymorphic DNAs (RAPD) amplified across 48 individuals from each parental species, and were transformed into 44 sequence-characterized amplified region (SCAR) markers. Five SNP markers that generated species-specific alleles for each species were selected from the 28 sequenced SCARs. A multiplex single nucleotide primer extension (SNuPE) analyses of the five SNPs using 40 A. mangium, 40 A. auriculiformis and 16 Acacia hybrids showed high discrimination power. This diagnostic system, with high discriminatory ability, provides a highly reliable and fast method for identifying interspecific hybrids of A. mangium and A. auriculiformis.