In this study, the incidence and genetic bases of nitrofurantoin resistance were established for clinical isolates of two successful clones of Salmonella enterica serovar Typhimurium, the pandemic "DT 104" and the pUO-StVR2 clone. A total of 61 "DT 104" and 40 pUO-StVR2 isolates recovered from clinical samples during 2008-2014 and assigned to different phage types, were tested for nitrofurantoin susceptibility. As previously shown for older isolates, all newly tested pUO-StVR2 isolates were highly resistant to nitrofurantoin (minimal inhibitory concentration [MIC] of 128 μg/ml), while 42.6%, 24.6%, and 32.8% of the "DT 104" isolates were susceptible, showed intermediate resistance or were highly resistant, with MICs of 8, 64, and 128 μg/ml, respectively. The genetic bases of nitrofurantoin resistance were established by PCR amplification and sequencing of the nfsA and nfsB genes encoding oxygen-insensitive nitroreductases. pUO-StVR2 isolates shared identical alterations in both nfsA (IS1 inserted into the coding region) and nfsB (in frame duplication of two codons). "DT 104" isolates with intermediate or high resistance had a missense mutation affecting the start codon of nfsA, while a single resistant isolate carried an additional frameshift mutation affecting nfsB. Complementation studies, performed with wild-type nfsA and nfsB, cloned independently and together into low and high copy-number vectors, confirmed NfsA and NfsB as responsible for nitrofurantoin toxicity. The same alterations persisted along time in isolates of each clone belonging to different phage types. Accordingly, changes leading to nitrofurantoin resistance have probably occurred before phage type diversification.