A method that combines fluorescence imaging and spectroscopy of single molecules at room temperature is presented. This approach allows us to identify a number of imaged molecules unequivocally by simultaneously recording their fluorescence emission spectra. Furthermore, their spectral characteristics not only allow us to separate different fluorescent labels quantitatively and qualitatively but also provide information on the microenvironment of the molecules. This new method was successfully tested on a system of yellow-green and red fluorescent 20-nm latex beads, and its usefulness in studies of biological systems was illustrated for a preparation of combined binary ratio labeling-fluorescence in situ hybridization-stained mouse chromosomes.