The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Lenin- grad/134/17/57 (H2N2) and B/USSR/60/69 were used to generate vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6 : 2 reassortant viruses containing the gene segments of the surface antigens haemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6 : 2 reassortant viruses are selected from co-infections between master donor virus and wild type viruses that the- oretically may yield as many as 256 combinations of gene segments and thus 256 enetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6 : 2 reassortant viruses are allowed as vaccine viruses, sboth selection and creening needs to be both rapid and unambiguous. The screen- ing of reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets is described to select both influenza A and influenza B 6 : 2 reassortant viruses to be used in seasonal and pan- demic live attenuated vaccine, unambigously. © 2011 Allerton Press, Inc.