A strategy directed at constructing polymorphic STSs from human chromosome 22 has yielded 15 poly(TG) microsatellite markers. A short insert plasmid library containing flow-sorted chromosome 22 DNA was screened with a labeled poly(AC) probe. A combination of sequencing techniques was used to identify the poly(TG) targets, primers were designed to flank these targets, and PCR screening was carried out on a panel of genomic and hybrid DNAs to determine heterozygosity and regional localization on chromosome 22. Twelve of the STSs are polymorphic. Markers with high heterozygosity have been localized to three subregions of 22q, with seven in the Giemsa-dark 22q12 band. The new chromosome 22 loci will be useful for mapping disease loci, for linkage analysis, and for PCR-based contig construction in the ongoing effort to map human chromosome 22.