This study was aimed to determine the prevalence of Rotaviruses in Karbala and Basrah provinces in Iraq, the genetic recombination of Rotavirus strains in human with another hosts and release a novel strains may lead limited activity of Rotarix vaccine. Rotaviruses were detected by Immunochromatography Test (ICT) then the positive samples were tested by reverse transcription PCR (RT-PCR) using specific primers to vp4gene, this gene is responsible for stimulation the immune system to produce neutralizing antibodies about of 200 diarrheal stool samples, 100 samples from pediatrics hospital of Karbala province and another 100 samples from Maternity and children hospital in Basrah province . The vp4 gene was implemented in rotavirus vaccine of Iraqi immunization program. The RT- PCR results showed that 56.3% (27/48) of samples positive in children under five years of age in Karbala province while Basrah province revealed 58.5% (31/53) positive samples in children. The results of sequencing of human and Rotarix vaccine revealed that there was relation between them and animal strains according to genetic reassortment between human and another species while in comparing with international strains there were closely related with Lebanon and Turkey human strains. Necessarily for further studies are needed to study the genetic alterations with viral recombination of rotavirus to detect the Rotarix vaccine activity against novel Rotaviral strains.