The development of a species‐specific marker for the analysis of the genetic polymorphism of the nitrogen‐fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal‐restriction fragment length polymorphism (T‐RFLP) methodology targeting specifically the 16S‐23S Ribosomal Intergenic Spacer of S. meliloti. Species‐specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T‐RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.