Short, tandemly repeated DNA motifs, termed SSRs (simple sequence repeats) are widely distributed throughout eukaryotic genomes and exhibit a high degree of polymorphism. The availability of size-based methods for genotyping SSRs has made them the markers of choice for genetic linkage studies in all higher eukaryotes, These genotyping methods are not efficiently applicable to mononucleotide repeats (MNRs). Consequently, MNRs, although highly frequent in the genome, have generally been ignored as genetic markers. In contrast to single nucleotide polymorphisms (SNPs), SSRs can be identified in silico once the genomic sequence or segment of interest is available, without requiring any additional information. This makes possible ad-hoc saturation of a target chromosomal region with informative markers. In this context, MNRs appear to have much to offer by increasing the degree of marker saturation that can be obtained. By using the human genome sequence as a model, computational analysis demonstrates that MNRs in the size of 9-15 bp are highly abundant, with an average appearance every 2.9 kb, exceeding di- and tri-nucleotide SSRs frequencies by two- and five-fold, respectively. In order to enable practical, high throughput MNR genotyping, a rapid method was developed, based on sizing of fluorescent-labeled primer extension products. Genotyping of 16 arbitrarily chosen non-coding MNR sites along human chromosome 22 revealed that almost two-thirds (63%) of them were polymorphic, having 2-5 alleles per locus, with 20% of the polymorphic MNRs having more than two alleles. Thus, MNRs have potential for in silico saturation of sequenced eukaryote genomes with informative genetic markers.