This document specifies a horizontal in vitro method for the molecular identification and differentiation of the monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium (1,4[5],12:i:-) lacking, or not expressing, the second H phase H:1,2, starting from isolates. The method detects specific DNA sequences of an intergenic region of the first H phase flagellin cluster for identification of Salmonella enterica subsp. enterica serovar Typhimurium (further called Salmonella Typhimurium) and specific DNA sequences of genes associated with second H phase flagellar antigen expression. The method is applicable for: differentiation of the isolate under analysis between monophasic Salmonella Typhimurium and the monophasic variant of another Salmonella non-Typhimurium serovar that has the same antigenic formula; identification of the isolate under analysis being either monophasic Salmonella Typhimurium or (biphasic) Salmonella Typhimurium. This document is applicable for the analysis of a pure culture belonging to the genus Salmonella, isolated from: products intended for human consumption; products intended for animal feeding; environmental samples in the area of food and feed production and handling; samples from the primary production stage. This document can also be applied in other domains for identification of monophasic Salmonella Typhimurium (e.g. environmental, human health, animal health).