The allele and haplotype frequencies for 13 Y-chromosome short tandem repeats (STRs) [nine STR loci of the minimal Y-chromosome haplotype (DYS19 -DYS385a -DYS385b -DYS389I -DYS389II -DYS390 -DYS391 -DYS392 -DYS393) plus four additional loci (DYS388, DYS426, DYS439, DXYS156)] were determined in 99 males from 4 Panamanian native American populations, including the Chibcha-speaking Ngöbé and Kuna and the Chocó-speaking Emberá and Wounan. Fifty haplotypes were identified, of which 48 (96%) were specific to a single population and 29 (63%) were found in only a single individual. Gene diversity per locus per population ranged from 0 to 0.814, with the highest gene diversity present at the DYS389II locus in the Emberá. The haplotypic discrimination capacity was low, ranging from 42.3% in the Kuna to 63.1% in the Wounan. The four tribes showed a high degree of differentiation both at the Y chromosome and in the mitochondrial genome, highlighting the importance of genetic structure even in geographically proximate and linguistically related populations.