Dissimilatory nitrate reduction to ammonia (DNRA) is the process in which nitrate is reduced, via nitrite, to ammonia. Bacteria known to carry out DNRA mainly originate from wastewater treatment plants, where DNRA is a relevant process. The ability to carry out DNRA is phylogenetically widespread, and the gene nrfA, encoding for the key enzyme of the second step of the pathway, could be used as a marker for this process. In this study we developed a new primer pair specific for nrfA in the genus Desulfovibrio. The specificity of the primer pair was tested on DNA from thirteen species of Desulfovibrio and DNA from two wastewater samples. PCR amplifications yielded products of the expected size (850 bp), and sequences obtained from Desulfovibrio strains and environmental sample clone libraries matched the Desulfovibrio nrfA gene. Nevertheless, we found nrfA gene sequences in the environmental samples that are not present in the databases. The new primer set can be used to obtain more sequences of the nrfA gene and improve our knowledge of the DNRA pathway in this genus, e.g. with the aim to improve the wastewater treatment process.