DNA-gold nanoparticle assemblies have shown promise as an alternative technology to DNA microarrays for DNA detection and RNA profiling. Understanding the effect of DNA sequences on the melting temperature of the system is central to developing reliable detection technology. We studied the effects of DNA base-pairing defects, such as mismatches and deletions, on the melting temperature of DNA-nanoparticle assemblies. We found that, contrary to the general assumption that defects lower the melting temperature of DNA, some defects increase the melting temperature of DNA-linked nanoparticle assemblies. The effects of mismatches and deletions were found to depend on the specific base pair, the sequence, and the location of the defects. Our results demonstrate that the surface-bound DNA exhibit hybridization behavior different from that of free DNA. Such findings indicate that a detailed understanding of DNA-nanoparticle assembly phase behavior is required for quantitative interpretation of DNA-nanoparticle aggregation.