Multiplex single nucleotide polymorphisms analysis has found a great demand in human genetics and pharmacogenetics. The present study reports a novel approach for a genotyping assay that could achieve simultaneous identification of multiple point mutations via a ligase-mediated gold nanoparticle assembly. Based on the allelic specificity of DNA ligase, gold nanoparticles modified by oligonucleotide probes perfectly matched to the DNA targets were assembled into a thermally-stable aggregate, while a single-base mismatch would result in the dissociation of the gold nanoparticle assembly at high temperature. Then, DNA targets and their point mutations could be differentiated using a multi-step temperature elevation analysis monitored by ultraviolet-visible measurements. This approach offered a direct colorimetric discrimination of multiple point mutations without stringent temperature control. The proposed approach is demonstrated using a model system for the identification of single-base mutations in codon 17 and position -28 of the beta-thalassemia gene. The results reveal that the wild and the mutant types could be simultaneously determined successfully. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in both research-oriented and clinical genomic assays.