Numerous nodule-specific genes, which are involved in the root nodule development and function, have been known and are still being discovered. Here, we reported the structure, expression, and genetic map location of two novel nodule-specific genes. First, two EST groups, one obtained from a nodule library and the other from all aboveground tissue libraries, were clustered with regard to in silico expression profiles. We compiled a pool of 103 putative nodule-specific sequence clusters. Then, two representative ESTs were selected for further experimental analyses. According to the full-length cDNA sequences, one was an EST of a novel nodule-specific polygalacturonase gene, GmPGN, and the other was an EST of a new short nodule-specific gene, GmEKN. The results of expression analyses of the GmPGN cDNAs indicated that GmPGN expression was not detectable in any of the soybean tissues except in the nodule tissue and may be regulated via alternative splicing. GmEKN expression was the most strongly detected in the nodule. The predicted GmEKN protein is both glutamic acid- and lysine-rich, and is also highly hydrophilic. Genetic mapping located GmPGN near the known quantitative trait locus conferring resistance to soybean cyst nematode on soybean molecular linkage group (MLG) B1, and GmEKN on MLG A2. These results provide useful information for the use of these genes in research on the orchestration of numerous genes in nodule development and function.