The study aims to assess the impact of rotavirus vaccine introduction on diarrheal diseases hospitalization and to identify the rotavirus genotypes most prevalent before and after vaccine introduction among children ≤ 5 years of age. Rotarix™ ® rotavirus vaccine is currently licensed for infants in Yemen and was introduced in 2012. The vaccination course consists of two doses. The first dose is administrated at 6 weeks of age and the second dose is completed by 10 weeks. Based on a longitudinal observational study, we assessed the impact of vaccination on rotavirus hospitalization before and after vaccination among children ≤ 5 years of age at the Yemeni-Swedish Hospital (YSH) in Taiz, Yemen. Prevaccination covered January 2009-July 2012 during which 2335 fecal samples were collected from children ≤ 5 years old. Postvaccination covered January 2013-December 2014 during which 1114 fecal samples were collected. Rotavirus was detected by Enzyme Linkage Immunosorbent Assay (ELISA). The incidence of rotavirus hospitalization decreased from 43.79% in 2009 to 10.54% in 2014. Hospitalization due to rotavirus diarrhea was reduced by 75.93%. Vaccine coverage increased from 23% in 2012 to 72% in 2014. Also, the results showed that the most predominant genotypes in prevaccination period were G2P[4] (55.0%), followed by G1P[8] (15.0%), while in postvaccination period G1P[8] (31%) was the predominant genotype, followed by G9P[8] (27.5%). In conclusion, rotavirus vaccination in Yemen resulted in sharp reduction in diarrheal hospitalization. A successful rotavirus vaccination program in Yemen will rely upon efficient vaccine delivery systems and sustained vaccine efficacy against diverse and evolving rotavirus strains.