The first genetic transcript map of the soybean genome was created by mapping one SNP in each of 1141 genes in one or more of three recombinant inbred line mapping populations, thus providing a picture of the distribution of genic sequences across the mapped portion of the genome. Singlenucleotide polymorphisms (SNPs) were discovered via the resequencing of sequence-tagged sites (STSs) developed from expressed sequence tag (EST) sequence. From an initial set of 9459 polymerase chain reaction primer sets designed to a diverse set of genes, 4240 STSs were amplified and sequenced in each of six diverse soybean genotypes. In the resulting 2.44 Mbp of aligned sequence, a total of 5551 SNPs were discovered, including 4712 single-base changes and 839 indels for an average nucleotide diversity of θ = 0.000997. The analysis of the observed genetic distances between adjacent genes vs. the theoretical distribution based upon the assumption of a random distribution of genes across the 20 soybean linkage groups clearly indicated that genes were clustered. Of the 1141 genes, 291 mapped to 72 of the 112 gaps of 5-10 cM in the preexisting simple sequence repeat (SSR)-based map, while 111 genes mapped in 19 of the 26 gaps >10 cM. The addition of 1141 sequence-based genic markers to the soybean genome map will provide an important resource to soybean geneticists for quantitative trait locus discovery and map-based cloning, as well as to soybean breeders who increasingly depend upon marker-assisted selection in cultivar improvement.