Resistance gene analogs (RGAs) characterized by the presence of nucleotide-binding sites (NBS) were cloned from <i>Vitis cinerea</i>, <i>V. rupestris</i>, and <i>V.</i> hybrid Horizon. Two degenerate PCR primer pairs were designed from conserved regions of NBS motifs within known resistance (<i>R</i>) genes and used for PCR amplification of putative RGAs. A total of 122 putative RGA sequences were cloned from all three genotypes by P-loop/GLPLAL-1 primers. Based on nucleic acid sequence-identity of 90% or greater, RGA clones were subdivided into eight, four, and seven groups for <i>V. cinerea</i>, <i>V. rupestris</i>, and Horizon, respectively. All of these clones showed similarity of nucleotide sequences to other known <i>R</i> genes or NBS-type nucleotide sequences, and seven clones showed high similarity. Thirty sequences were cloned from <i>V. cinerea</i> by P-loop/Rev loop and subdivided into four sequence groups, none of which were similar to nucleotide sequences of other <i>R</i> genes. Nineteen representative RGA clones were classified into 13 TIR- (<i>Drosophila</i><i>T</i><i>oll</i> and mammalian <i>Interleukin-1</i><i>R</i><i>eceptors</i>) NBS-leucine rich repeat (LRR)-like genes and six non-TIR-NBS-LRR-like genes based primarily on nucleotide sequences of kinase-2 motifs and phylogenetic analysis with known TIR or non-TIR proteins. Twenty-three sequence tagged site (STS) and three cleaved amplified polymorphic sequence (CAPS) markers developed from RGAs were checked for segregation among 179 seedlings from Horizon x Illinois (Ill.) 547-1, and 18 showed goodness-of-fit using a chi-square test. Marker stkVa011 correlated with segregation for downy mildew resistance in this population. These STS markers are currently being investigated for their potential in molecular breeding for disease resistance.