Background During the course of development and characterization of various rotavirus reassortants, we found that the relative position of the gene encoding neutralization and protective antigen VP7 of certain rotavirus strains in a PAGE gel was influenced by the concentration of acrylamide. Objectives We investigated systematically various factors that affected the relative position of the rotavirus VP7 gene in a PAGE gel. Study design We analyzed dsRNAs of selected rotavirus strains bearing G1, G2, G3 or G9 specificity by PAGE at varying concentrations of acrylamide. Results We demonstrated that the relative position of the VP7 gene of three G2 strains varied depending upon the concentration of acrylamide in a PAGE gel, which occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus gene background; and the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this phenomenon when the PAGE running conditions were varied. Conclusions The concentration of acrylamide in a PAGE gel was the major factor that influenced the relative position of the VP7 gene of G2 rotavirus strains (i.e., VP7 gene coding assignment by PAGE).