A new fluorescence-based method for inbred haploid differentiation in maize kernels was developed by utilizing the R1-nj color marker in combination with fluorescence micro-spectroscopy and imaging. Seven inbred lines with varying R1-nj expression were used in this study. The fluorescence response of the diploid kernels at the embryonic dye spot was shown to simultaneously exhibit lower intensity and occur at a higher wavelength than the fluorescence of the dye-lacking haploid embryos. Intensity and area thresholds were applied to fluorescence images to sort the haploids from mixed sample populations, and sorting efficiencies of greater than 80% were achieved in all seven inbred lines (with values greater than 90% for five lines). The potential for high throughput sorting when fluorescence imaging is combined with existing technologies for seed handling as well as high sorting efficiency may make fluorescence a viable and promising alternative to current sorting methods for some inbred lines.