Infection with different strains of Helocobacter pylori and emergence of new genomic variants during their long-term gastric colonization are assumed to be the main reasons for eradication failure. We used genotyping and arbitrarily primed PCR fingerprinting (RAPD) to detect relatedness and genetic variations among H. pylori single isolates from each patient in Iran. Multiplex-PCR amplification of gene alleles encoding the virulence factors vacA (m/s), cagA, and iceA (A1/A2) and comparison of RAPD patterns of different singles colonies were performed for each individual patient's isolate. Results showed a high frequency of diversity among the H. pylori strains. Nearly 23% of infected patients showed a single type infection. The infection types related, unrelated, and related/unrelated were found among 25.6%, 12.8%, and 38.5% of patients, respectively. Both mixed type infections (77%) and quasispecies development (15.4%) were detected in these patients. Genotype conversion among vacA (41.6%), cagA (41.6%), and iceA (50%) alleles was observed for the strains with identical or related RAPD patterns. Coevolution of different alleles was also detected in a patient infected with strains presenting the same RAPD patterns. Collectively, results of this study revealed the occurrence of quasispecies development, mixed type infections, and changes in virulence properties of H. pylori strains among the studied patients.