Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.