Single nucleotide polymorphisms (SNPs) represent the most prevalent class of genetic markers available for genetic linkage analysis. Detection of SNPs using fluorescently tagged primers offers a new method for screening a population for polymorphic markers. In this report we describe the use of PCR primers in detection of single nucleotide polymorphisms based upon 3' mismatch and allele-specific amplification of preamplified target sequences. A sequence library was generated from multiple barley cultivars, then screened for polymorphisms between cultivars. From polymorphic sequences between cultivars, two primers were developed with different phlourophores attached on the 5' end of the primer and the polymorphic nucleotide at the 3' end. The fluorescently labeled primers are then used in a subsequent PCR reaction. The genotypic differentiation of this specificity of this PCR is based upon 3' specific annealing of the primer at the site of polymorphism.