Pediatric acute respiratory tract infections (ARTIs) are a leading cause of morbidity and mortality. The objectives of this study were to describe the epidemiology of polymicrobial ARTI in a hospital-based pediatric population and to investigate the association of polymicrobial infection and severity of illness. We conducted a retrospective study of 559 archived respiratory specimens from 421 children under the age of 10 years collected from March 28, 2008 through June 30, 2009 and stored by the University of Iowa Hospital and Clinics Clinical Microbiology Laboratory. Specimens were tested by immunofluorescence assay and/or viral culture at the time of collection (influenza A and B, parainfluenza [PIV] 1-3, respiratory syncytial virus [RSV], adenovirus [Ad]) and uniformly by RT-PCR (human metapneumovirus [hMPV], rhinovirus [HRV], human bocavirus [HBoV]) and PCR (Ad) for the current study. Demographic and clinical data were abstracted from electronic medical records. Results from this study suggest that polymicrobial respiratory tract infections are common in this population. A virus was identified in 61.3% of 349 respiratory specimens from children with confirmed or suspected ARTI. HRV (27.5%), RSV (18.9%), HBoV (8.3%), hMPV (7.7%), and PIV (6.6%) were the most common viruses detected. A viral coinfection was identified in 21.5% of the 214 virus-positive specimens and was most often detected for Ad (53.3% of 15 Ad-positive specimens), HBoV (51.7% of 29 HBoV-positive specimens), PIV (43.5% of 23 PIV-positive specimens), HRV (35.4% of 96 HRV-positive specimens), and RSV (34.8% of 66 RSV-positive specimens). Among the 46 specimens with dual or triple viral coinfections detected, the most frequent virus-virus combination was HRV-RSV (n=12). We hypothesized that certain host-specific risk factors were associated with the likelihood of viral coinfection. While none of the covariates in the final model were significant, the results were suggestive. Male gender (OR 1.70, 95% CI 0.83-3.46), age between 6 months to 1 year (as compared to children less than 6 months old, OR 2.15, 95% CI 0.75-6.19), and history of any chronic condition that may result in immunosuppression (OR 2.05, 95% CI 0.99-4.23) were each associated with increased odds of viral coinfection (p > 0.05). We also hypothesized that children with coinfections would be more likely to have severe ARTI. Children with viral-bacterial coinfection, as compared to children with viral mono-infection, were more likely to be admitted to an intensive care unit (OR 6.00, 95% CI 2.51-14.33) even after controlling for age, history of prematurity, urban/rural residence, and leukocytosis. This study will inform medical and public health professionals with regard to the epidemiology of polymicrobial infections and their potential importance as a cause of severe acute respiratory tract infection in children. Furthermore, results of this study may contribute to the ongoing discussion of the importance of diagnostic ability to reliably detect multiple concurrent pathogens in a single individual.