Esophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana, where Necator americanus (human hookworm) also exists at high prevalence. Yet, very little is known about the transmission patterns of O. bifurcum, which is in part due to the difficulties in diagnosis and in differentiating some life-cycle stages of O. bifurcum from N. americanus using morphological features. As a first step toward developing a molecular-diagnostic assay, it was evaluated whether ribosomal (r)DNA could provide genetic markers for the identification of O. bifurcum and N. americanus to species. Internal transcribed spacer rDNA (plus flanking and intervening sequences) was analyzed by polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) using several restriction endonucleases. The analysis showed that there was no detectable intraspecific difference in the size of the PCR products among multiple samples, that there was a consistent size difference in the products (of 110 bp or 350 bp, depending on region amplified) between the species, and that there was no significant variation in restriction patterns within each species. These results indicate that the rDNA spanning the internal transcribed spacers provides useful genetic markers for the identification of O. bifurcum and N. americanus to species, which has important implications for developing PCR-based tools to study the epidemiology and population biology of O. bifurcum.