Seventeen Y-chromosomal short tandem repeat loci were analyzed in a sample of 435 unrelated healthy male individuals from Southern Poland (including highlanders from Tatra Mountains). One duplication in the locus DYS389II (29,30) and five microvariant alleles in the locus DYS458 were found. The most frequent haplotype, found in three individuals, was as follows (in the order of Yfiler loci): {16, 13, 25, 30, 15, 15, 11/14, 13, 11, 11, 10, 23, 11, 13, 14, 11, 21}. Gene diversity for South Poland population amounts close to 1.000. Performed differentiation test between all pairs of samples, based on haplotype frequencies, represented as non-differentiation exact P values indicates that there is no statistically significant differences in haplotype frequencies between South Poland and Austrian as well as South Poland and Wallachian populations.