In Spain, diarrhea remains a major cause of illness among infants and young children. To determine the prevalence of rotavirus genotypes and temporal and geographic differences in strain distribution, a structured surveillance study of hospitalized children <5 years of age with diarrhea was initiated in different regions of Spain during 2005. Rotavirus was detected alone in samples from 362 (55.2%) samples and as a coinfection with other viruses in 41 samples (6.3%). Enteropathogenic bacterial agents were detected in 4.9% of samples; astrovirus and norovirus RNA was detected in 3.2% and 12.0% samples, respectively; and adenovirus antigen was detected in 1.8% samples. Including mixed infections, the most predominant G type was G9 (50.6%), followed by G3 (33.0%) and G1 (20.2%). Infection with multiple rotavirus strains was detected in >11.4% of the samples studied during 2005.