Each year, billions of day-old layer chicks are produced in the world. Since only female chicks are reared for egg production, the chicks must be sexed and the unwanted male layer chicks are culled. The culling of male chicks is a serious problem, both in terms of animal welfare and waste disposal. The germinal disc in fertilized but unincubated eggs contains already several thousands of blastoderm cells. The cellular DNA in birds is different for male and female chicks. The difference in DNA content between male and female chicks is around 2% and is measurable by Fourier transform infrared (FT-IR) spectroscopy. In this study, small amounts of blastoderm cells from 22 chicken eggs were characterized by attenuated total reflection FT-IR spectroscopic imaging and classified by linear discriminant analysis. Polymerase chain reaction (PCR) was used as a reference method to determine the gender. The spectroscopic results demonstrate that male blastoderm cells exhibit a higher content of DNA than cells from female blastoderm. The spectroscopic-based gender determination led to the same result as the PCR analysis. FT-IR spectroscopic imaging allows the gender determination of unincubated eggs within a few seconds based on the accurate determination of the different DNA contents in blastoderm cells of both sexes.