In 2007, three strains of Salmonella enterica serotype Rissen (S. Rissen) were isolated in the laboratory of diagnostic microbiology of the General Hospital of Prato, Tuscany, Italy, over a 1 month and half interval of time. The first isolate was recovered on January 26 from an outpatient with enteritis. Then, two strains were isolated on February 16 and March 11 respectively, from central venous catheters of patients who were being hospitalized in two departments of the Hospital. An epidemiologically linked cluster of cases of salmonellosis was suspected. The three strains were submitted to single enzyme-amplified fragment length polymorphism (SE-AFLP) and XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE) that yielded undistinguishable profiles. Epidemiological investigations failed to identify a common source of infection within the Hospital. Moreover, the third patient had been exclusively total parenteral nutrition fed since his admission with a stomach cancer diagnosis. The first patient had a community-acquired infection, but the source of her illness was uncertain. Twenty-five further isolates identified in the years 2004–2007 in the same geographical area showed distinctly different PFGE and SE-AFLP patterns. The three patients seemed to represent a cluster of epidemiologically unrelated cases caused by a previously never recognized S. Rissen strain. Rapid subtyping of isolates is essential in the early investigation of potential outbreaks, but synthesis of conventional and molecular epidemiological investigation and availability of surveillance data is often critical to prevent the initiation of time-consuming, expensive and ineffective further investigations and control interventions.