We previously reported non-random selections of human rotavirus (HRV) Wa genes 2 and 5 in reassortant formation between HRV strains Wa and HN126 under selection pressure with neutralizing monoclonal antibodies. In order to study whether or not these genes are preferentially selected in the genetic background of a parental strain HN126 in vitro without selection pressures, coinfection and multiple passage experiments were performed between HN126 and one of three reassortants, C1, C1T and C1F; C1 possessed genes 2 and 5 derived from Wa and the other genes derived from HN126, while C1T and C1F were single gene reassortants having Wa gene 2 or Wa gene 5 in the genetic background of HN126, respectively. When MA-104 cells were coinfected with the same infectious units of HN126 and C1, Wa genes 2 and 5 of reassortant C1 became predominant within 10 repeated passages, although Wa gene 5 was selected more preferably than Wa gene 2. Similar results were obtained under different experimental conditions in which different doses of parental strains or different type of cells were used. Also, in coinfections of MA-104 cells with HN126 and C1T, or HN126 and C1F, Wa gene 2 or Wa gene 5 became predominant at the sixth passage. Analysis of viral growth curves indicated that two reassortants, C1 and C1F, replicated to a titre higher than HN126, while no difference in viral growth was observed between C1T and HN126. These results indicated that in the genetic background of HN126, Wa gene 5 might provide viruses with a growth advantage compared with its HN126 counterpart, while Wa gene 2 might be preferentially selected into reassortant clones through its greater functional capacity for assortment during viral replication.