The melting temperature of probe-target nucleic acid hybrids plays an important role in sensor performance. This study employed surface plasmon resonance (SPR) techniques to investigate the hybridization of both DNA and RNA targets to their complementary surface-bound DNA oligonucleotide and the corresponding melting temperatures. The target molecule was a 26 nucleotide strand of RNA selected because of its utility in detecting the tobacco mosaic virus, a common plant pathogen with an RNA genome. The melting temperatures of duplexes with immobilized probes were determined by hybridization of the DNA and RNA targets with the bound DNA probe at different temperatures using a custom-built thermostated dual-channel SPR cell. Hybridization conditions and binding efficiencies were compared for both DNA and RNA binding to the gold-coated surface-bound DNA probe. The melting temperature for the DNA-DNA duplex was approximately 15. °C higher than the DNA-RNA in the solid state. The melting temperature in solution was also measured for comparison to the surface values. For both RNA and DNA, the melting transitions were substantially lower (~20 °C) for solid state binding relative to solution. Particularly for surface immobilized DNA-RNA hybrids, the melting temperature was reduced to room temperature or below. Since most biosensor platforms operate at room temperature, they may consequently exhibit poor sensor performance due to weakened probe-target interactions. This result emphasizes that for optimal sensor performance, the hybrid melting temperature should be considered. The ability to study and optimize the binding of complementary strands of genomic materials to bound DNA probes could facilitate the rapid detection and identification of plant viruses having genomic RNA using various biosensor rapid analytical techniques.