To investigate the microevolution of Helicobacter bizzozeronii in the human stomach, comparative genomics of antrum-derived populations, obtained 3 months before (T(0)) and 6 months after (T(1)) an unsuccessful eradication treatment, was performed. For each time point, the DNA of bacterial mass, representing the population diversity in three biopsies, was mixed in equal amounts and sequenced using Illumina technology. Polymorphic sites (PSs) were detected by mapping the reads against an isogenic reference genome, derived from a corpus isolate obtained at T(0). The total numbers of PSs detected in the H. bizzozeronii population at T(0) and T(1) were 128 and 223, affecting 81 and 134 coding sequences, respectively. At T(0) in 91.4% of the PSs the mutation appeared at a frequency of 50% or less. On the contrary, in the majority of the PSs observed in T(1) (71.3%) the mutation had a frequency >75%. Although only a minority of mutations were fixed in the antrum-derived population at T(0), a certain level of allelic variability, compared with the corpus-derived reference genome, was present and most likely arose as consequence of the long-term colonization of the patient. The treatment probably induced a sudden decrease of population size, selecting a subpopulation, which acted as founder for the new population at T(1) characterized by a higher number of fixed mutations. These data demonstrate that genome plasticity is an important common prerequisite among gastric Helicobacter species for adaptation to the stomach environment allowing the bacterium to evolve rapidly once a selective pressure is applied.