OBJECTIVE - To evaluate two saliva collection methods for DNA yield and quality as applied to a large, integrated, multicentre, European project involving the collection of biological material from children. DESIGN - Cross-sectional multicentre comparative study in young children. METHODS - Saliva samples were collected from 14,019 children aged 2-9 years from eight European countries participating in the IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) study. This involved either the collection of 2 ml of saliva from children who were able to spit, or using a sponge to collect whole saliva and buccal mucosal cells from the inside of the mouth of younger children unable to spit. Samples were assembled centrally in each participating centre and subsequently despatched for DNA extraction and biobanking to the University of Glasgow. A subgroup of 4678 samples (∼33% of sampled individuals) were chosen for DNA extraction before genotyping. RESULTS - The whole-saliva collection method resulted in a higher DNA yield than the sponge collection method (mean±s.d.; saliva: 20.95±2.35 μg, sponge: 9.13±2.25 μg; P<0.001). DNA quality as measured by A (260)/A (280) was similar for the two collection methods. A minimum genotype calling success rate of 95% showed that both methods provide good-quality DNA for genotyping using TaqMan allelic discrimination assays. CONCLUSIONS - Our results showed higher DNA yield from the whole-saliva collection method compared with the assisted sponge collection. However, both collection methods provided DNA of sufficient quantity and quality for large-scale genetic epidemiological studies.