Background: The increasing incidence of Clostridium difficile infection (CDI) in hospitalised paediatric populations, combined with the emergence of hypervirulent strains, community acquired CDI and the need for prompt treatment and infection control measures, makes the rapid, accurate diagnosis of CDI crucial. Few studies have validated available methods in a paediatric population. Aim: To validate commonly used C. difficile diagnostic tests in a paediatric hospital population. Methods: From October 2011 to January 2012 150 consecutive stools from 75 patients were collected at a tertiary paediatric hospital laboratory in Perth, Western Australia. Each stool was tested using: C. Diff Quik Chek Complete (QCC) (detecting glutamate dehydrogenase and toxin A/B) (Techlab), Illumigene C. difficile (detecting tcdA) (Meridian Bioscience), BD GeneOhm Cdiff (detecting tcdB) (Becton Dickinson) and two culture methods: cycloserine cefoxitin fructose agar (CCFA) and cell cytotoxin neutralisation assay (CCNA) direct from stool and Cdiff Chromagar (BioMérieux) and an in house real time PCR on isolates for tcdA, tcdB, cdtA, and cdtB. Isolates were ribotyped. Results: Forty-three percent of patients were C. difficile culture positive. All methods demonstrated excellent specificity (97-100%). Chromagar and genotyping had the highest sensitivity (94%), higher than CCFA (85%) and CCNA (33%). QCC GDH had a NPV of 93% but toxin A/B sensitivity was poor (29%). Sensitivities were similar for the BD GeneOhm (91%) and Illumigene (89%). Algorithms using GDH first followed by a molecular test reduced the sensitivity to 85% for both Illumigene and BD GeneOhm. One ribotype predominated – UK14/20 (27%). Conclusion: Commonly used C. difficile diagnostic tests have similar characteristics in a paediatric and adult populations. Toxin EIA testing should not be used in isolation. In this study a GDH first algorithm reduced the sensitivity of the confirmatory test and, given the excellent sensitivity of the molecular methods, these are recommended over the GDH, culture and genotyping/CCNA methods.