The ability to identify specific chromosomes with certainty has been established by the development of several cytogenetic techniques based on staining. Here, we report the use of a new optical technique, laser tweezers and Raman spectroscopy (LTRS), to capture and manipulate chromosomes in order to obtain their spectral patterns for molecular analysis without the need for staining. The purpose of this study was to obtain Raman spectroscopy patterns for chromosomes number 1, 2, and 3 and to test if the Raman spectroscopy pattern could be used to distinguish these three chromosomes. In our experiment, optical tweezers were used to capture the individual chromosomes and the Raman spectral patterns were collected for the trapped chromosomes. Then, the captured chromosome was manipulated with the optical tweezers and moved to another chamber through a micro - channel, in which the chromosomes were G-banded for positive identification as chromosome number 1, 2, or 3. Generalized discriminate analysis (GDA) was used to compare the Raman signatures. This analysis revealed that chromosomes 1, 2, and 3 could be distinguished and identified based on their Raman spectra. Development of this approach will lead to more rapid automatic methods for chromosome analysis and identification without the use of prior staining. Moreover, the Raman spectral patterns may lend themselves to more detailed analysis of chromosomal structure than is currently available with standard staining protocols. Such analysis may some day be useful for rapid, automated screening and diagnosis for certain cancers. © 2006 Optical Society of America.