The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, based on peptide-like backbone. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences, and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double stranded DNA, the unique physico-chemical properties of PNAS have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy, genome mapping and mutation detection. Over the last few years, the use of PNAS has proven its powerful usefulness in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, spermatozoa as well as on isolated oocytes and blastomeres. Muticolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful complement to FISH for in situ chromosomal investigation.